Carrier-Linked Treprostinil Prodrugs

ABSTRACT

The present invention relates to prodrugs or a pharmaceutically acceptable salt thereof comprising a covalent treprostinil carrier conjugate as well as pharmaceutical composition comprising said compounds. The compounds may be used as medicaments, especially for diseases or disorders which can be treated by treprostinil, such as pulmonary arterial hypertension (PAH).

The present application is a continuation of U.S. patent applicationSer. No. 14/238,437 filed on Feb. 11, 2014, which claims priority fromPCT Patent Application No. PCT/EP2012/065745 filed on Aug. 10, 2012,which claims priority from European Patent Application No. EP 11177409.7filed on Aug. 12, 2011, European Patent Application No. EP 11195615.7filed on Dec. 23, 2011, and European Patent Application No. EP12165508.8 filed on Apr. 25, 2012 the disclosures of which areincorporated herein by reference in their entirety.

FIELD OF THE INVENTION

It is noted that citation or identification of any document in thisapplication is not an admission that such document is available as priorart to the present invention.

Pulmonary arterial hypertension (PAH) is an increase in blood pressurein the pulmonary artery, pulmonary vein, or pulmonary capillaries,leading to shortness of breath, dizziness, fainting, and other symptoms,all of which are exacerbated by exertion. PAH can be a severe diseasewith a markedly decreased exercise tolerance and heart failure. It is anorphan disease with an incidence of about 2-3 per million per year and aprevalence of about 15 per million. Median survival of patients withuntreated PAH is in the range of 2-3 years from time of diagnosis, withthe cause of death usually being right ventricular failure.

Pulmonary arterial hypertension involves the vasoconstriction ortightening of blood vessels connected to and within the lungs. Overtime, fibrosis causes the affected blood vessels to become both stifferand thicker which further increases the blood pressure within the lungsand impairs their blood flow. In addition, the increased workload of theheart causes hypertrophy of the right ventricle which ultimately causesright heart failure. As the blood flowing through the lungs decreases,the left side of the heart receives less blood and thus oxygen supply isbelow the required level, especially during physical activity.

A number of agents have been introduced for the treatment of PAH ofwhich prostacyclins are commonly considered to be the most effective.One prostacyclin is Epoprostenol which is a synthetic prostacyclin andmarketed as Flolan® (GlaxoSmithKline). It is given to patients viacontinuous infusion and requires a semi-permanent central venouscatheter which can cause sepsis and thrombosis. Flolan® is unstable, andtherefore has to be kept on ice during administration. Since it has ahalf-life of only 3 to 5 minutes, the infusion has to be continuousnight and day and any interruption can be fatal. Thus, treatment of PAHwith Flolan® is a huge burden for the patient.

Another prostacyclin, Iloprost (Ilomedin) which is marketed as Ventavis®(Bayer), was the only inhaled form of prostacyclin approved for use inthe US and Europe, until the inhaled form of treprostinil was approvedby the FDA in July 2009 which is marketed under the trade name TYVASO®(United Therapeutics).

Inhaled prostacyclin suffer from the drawback of not providing fullyefficacious plasma levels of drug throughout the dosing period, makinginhaled therapy less desired in severe patients.

Therefore, there was a need to develop other prostanoids, as has beendescribed for example in U.S. Pat. No. 4,306,075A and EP159784B1. Onesuch prostaglandin is treprostinil with the trade name Remodulin®(United Therapeutics). The half-life of treprostinil is 4 hours buttreprostinil is still required to be administered as a continuoussubcutaneous infusion or continuous intravenous infusion via an infusionpump that the patient must wear at all times.

Subcutaneous infusion of treprostinil is frequently painful to theextent that the patient cannot tolerate the pain and consequently themode of administration is switched to intravenous infusion. However, anincreased risk of sepsis with intravenous Remodulin has been reported.

As subcutaneous infusion is associated with pain, there is a need fordeveloping a prostacyclin that can be administered by subcutaneousadministration but with reduced rates of pain. This can be achieved byadministering a carrier linked prodrug of a prostacyclin, in which theabsorption of the prodrug is sufficiently fast, and release ofprostacyclin from the prodrug is sufficiently slow, such thesubcutaneous exposure to free prostacyclin molecules is minimized.

Prostacyclins are the standard treatment of PAH, particularly in moresevere patients. Although inhaled treprostinil is more convenient andwithout the strong pain that is frequently associated withsubcutaneously infused treprostinil, inhalation is considered to be lesseffective and therefore less often prescribed.

Treprostinil has the following structure:

Therefore, there exists a need to provide a more efficacious and/or morecomfortable treprostinil treatment for patients.

It is noted that in this disclosure and particularly in the claimsand/or paragraphs, terms such as “comprises”, “comprised”, “comprising”and the like can have the meaning attributed to it in U.S. Patent law;e.g., they can mean “includes”, “included”, “including”, and the like;and that terms such as “consisting essentially of” and “consistsessentially of” have the meaning ascribed to them in U.S. Patent law,e.g., they allow for elements not explicitly recited, but excludeelements that are found in the prior art or that affect a basic or novelcharacteristic of the invention.

It is further noted that the invention does not intend to encompasswithin the scope of the invention any previously disclosed product,process of making the product or method of using the product, whichmeets the written description and enablement requirements of the USPTO(35 U.S.C. 112, first paragraph) or the EPO (Article 83 of the EPC),such that applicant(s) reserve the right to disclaim, and herebydisclose a disclaimer of, any previously described product, method ofmaking the product, or process of using the product.

SUMMARY OF THE INVENTION

This object is achieved with carrier-linked treprostinil prodrugs offormula (I):

Z¹-X⁰—T)_(y)  (I),

wherein each T is independently selected from structures (i) to (v):

-   -   wherein dashed lines indicating attachment to X⁰;

y is an integer ranging of from 1 to 64, preferably ranging from 1 to16, more preferably y selected from 1, 2, 3, 4, 5, 6, 7 8, 9, 10, 11,12, 13, 14, 15 and 16, even more preferably y is 4, 6, or 8, mostpreferably, 8,

each X⁰ is independently (X^(0A))_(m1)-(X^(0B))_(m2);

m1; m2 are independently 0; or 1;

X^(0A) is T⁰;

X^(0B) is a branched or unbranched or cyclic C₁₋₁₅ alkylene group whichis unsubstituted or substituted with one or more R³, which are the sameor different; more preferably, X^(0B) is a branched or unbranched cyclicC₁₋₁₅ alkylene group which is unsubstituted or substituted with one ormore R³, which are the same or different

R³ is halogen; C₁₋₆alkyl; CN; C(O)R⁴; C(O)OR⁴; OR⁴; C(O)R⁴;C(O)N(R⁴R^(4a)); S(O)₂N(R⁴R^(4a)); S(O)N(R⁴R^(4a)); S(O)₂R⁴; S(O)R⁴;N(R⁴)S(O)₂N(R^(4a)R^(4b)); SR⁴; N(R⁴R^(4a)); NO₂; OC(O)R⁴;N(R⁴)C(O)R^(4a); N(R⁴)SO₂R^(4a); N(R⁴)S(O)R^(4a);N(R⁴)C(O)N(R^(4a)R^(4b)); N(R⁴)C(O)OR^(4a); OC(O)N(R⁴R^(4a)); or T⁰;

R⁴, R^(4a), R^(4b) are independently selected from the group consistingof H; T⁰; C₁₋₆ alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl, wherein C₁₋₆alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl are optionally substituted withone or more R⁵, which are the same of different;

R⁵ is halogen; CN; C(O)R⁶; C(O)OR⁶; OR⁶; C(O)R⁶; C(O)N(R⁶R^(6a));S(O)₂N(R⁶R^(6a)); S(O)N(R⁶R^(6a)); S(O)₂R⁶; S(O)R⁶;N(R⁶)S(O)₂N(R^(6a)R^(6b)); SR⁶; N(R⁶R^(6a)); NO₂; OC(O)R⁶;N(R⁶)C(O)R^(6a); N(R⁶)SO₂R^(6a); N(R⁶)S(O)R^(6a);N(R⁶)C(O)N(R^(6a)R^(6b)); N(R⁶)C(O)OR^(6a); OC(O)N(R⁶R^(6a));

R⁶, R^(6a), R^(6b) are independently selected from the group consistingof H; C₁₋₆ alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl, wherein C₁₋₆ alkyl;C₂₋₆ alkenyl; and C₂₋₆ alkynyl are optionally substituted with one ormore halogen, which are the same of different;

T⁰ is phenyl; naphthyl; azulenyl; indenyl; indanyl; C₃₋₇ cycloalkyl; 3to 7 membered heterocyclyl; or 8 to 11 membered heterobicyclyl, whereinT⁰, is optionally substituted with one or more R⁷, which are the same ordifferent;

R⁷ is halogen; CN; COOR⁸; OR⁸; C(O)R⁸; C(O)N(R⁸R^(8a));S(O)₂N(R⁸R^(8a)); S(O)N(R⁸R^(8a)); S(O)₂R⁸; S(O)R⁸;N(R⁸)S(O)₂N(R^(8a)R^(8b)); SR⁸; N(R⁸R^(8a)); NO₂; OC(O)R⁸;N(R⁸)C(O)R^(8a); N(R⁸)S(O)₂R^(8a); N(R⁸)S(O)R^(8a); N(R⁸)C(O)OR^(8a);N(R⁸)C(O)N(R^(8a)R^(8b)); OC(O)N(R⁸R^(8a)); oxo (═O), where the ring isat least partially saturated; C₁₋₆ alkyl; C₂₋₆ alkenyl; or C₂₋₆ alkynyl,wherein C₁₋₆ alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl are optionallysubstituted with one or more R⁹, which are the same or different;

R⁸, R^(8a), R^(8b) are independently selected from the group consistingof H; C₁₋₆ alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl, wherein C₁₋₆ alkyl;C₂₋₆ alkenyl; and C₂₋₆ alkynyl are optionally substituted with one ormore R¹⁰, which are the same of different;

R⁹, R¹⁰ are independently selected from the group consisting of halogen;CN; C(O)R¹¹; C(O)OR¹¹; OR¹¹; C(O)R¹¹; C(O)N(R¹¹R^(11a));S(O)₂N(R¹¹R^(11a)); S(O)N(R¹¹R^(11a)); S(O)₂R¹¹; S(O)R¹¹;N(R¹¹)S(O)₂N(R^(11a)R^(11b)); SR¹¹; N(R¹¹R^(11a)); NO₂; OC(O)R¹¹;N(R¹¹)C(O)R^(11a); N(R¹¹)SO₂R^(11a); N(R¹¹)S(O)R^(11a);N(R¹¹)C(O)N(R^(11a)R^(11b)); N(R¹¹)C(O)OR^(11a); and OC(O)N(R¹¹R^(11a));

R¹¹, R^(11a), R^(11b) are independently selected from the groupconsisting of H; C₁₋₆ alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl, whereinC₁₋₆ alkyl; C₂₋₆ alkenyl; and C₂₋₆ alkynyl are optionally substitutedwith one or more halogen, which are the same of different;

Z¹ is a carrier comprising a covalently bound polymer, preferably apharmaceutically acceptable polymer,

wherein the carrier is covalently attached to a moiety X⁰, provided thatone of m1, m2 is 1 and wherein the carrier is covalently attached to Tin case m1, m2=0,

or a pharmaceutically acceptable salt thereof.

It was surprisingly found that such carrier-linked treprostinil prodrugscan be used to obtain dosage forms of treprostinil which at leastpartially overcome the above mentioned shortcomings.

DETAILED DESCRIPTION OF EMBODIMENTS

It is to be understood that the figures and descriptions of the presentinvention have been simplified to illustrate elements that are relevantfor a clear understanding of the present invention, while eliminating,for purposes of clarity, many other elements which are conventional inthis art. Those of ordinary skill in the art will recognize that otherelements are desirable for implementing the present invention. However,because such elements are well known in the art, and because they do notfacilitate a better understanding of the present invention, a discussionof such elements is not provided herein.

The present invention will now be described in detail on the basis ofexemplary embodiments.

Within the present invention the terms are used having the meaning asfollows.

The terms “drug”, “biologically active molecule”, “biologically activemoiety”, “biologically active agent”, “active agent”, “active substance”and the like mean any substance which can affect any physical orbiochemical properties of a biological organism, including but notlimited to viruses, bacteria, fungi, plants, animals, and humans. Inparticular, as used herein, the terms include any substance intended fordiagnosis, cure, mitigation, treatment, or prevention of disease inorganisms, in particular humans or other animals, or to otherwiseenhance physical or mental well-being of organisms, in particular humansor animals. Treprostinil is understood to be a biologically activemolecule.

“Biologically active moiety D” means the part of the drug linkerconjugate, which results after cleavage in a drug D-OH or D-H of knownbiological activity. Accordingly, “biologically active moietytreprostinil” or “treprostinil moiety” means the part of thecarrier-linked treprostinil prodrug, which results after cleavage in adrug treprostinil of known biological activity.

“Free form” of a drug such as treprostinil refers to the drug in itsunmodified, pharmacologically active form, such as after being releasedfrom a carrier-linked prodrug.

Targeting moieties are moieties that when present in a molecule, such asfor example a prodrug, allow preferential localization of such largermolecule in specific target areas of the organism to which it has beenadministered. Such specific target areas might be organs, certain celltypes or subcellular compartments. “Preferential localization” meansthat at least 5%, 10%, preferably at least 20% and more preferably atleast 30% of the biologically active moieties administered to a patientreach said specific target areas.

Targeting moieties may be divided into 3 classes according to size:

-   -   small molecular targeting moieties, for example C-glucuronide,        cobalamin, vitamins such as folic acid (folate) and analogs and        derivatives, carbohydrates, bisphosphonates,        N-acetylgalactosamine,    -   peptides, for example bombesin, somatostatin, LHRH, EGF, VEGF,        hCG, fragments of luteinizing hormone (LH), Octreotide,        Vapreotide, Lanreotide, RC-3940 series, Decapeptyl, Lupron,        Zoladex, Cetrorelix, peptides or peptidomimetics containing the        NGR or RGD motifs or derived from these motifs such as CNGRC        (linear), GNGRG (cyclic), ACDC RGD CFCG (cyclic), CDCRGDCFC,        CNGRC (cyclic), CRGDCGG, CNGRC, or other peptides such as        ATWLPPR, Thrombospondin (TSP)-1 mimetics, (RGD peptidomimetic),        CTTHWGFTLC, CGNKRTRGC, neuropeptide substance P, SSP, the Sar9,        Met(O2)11 analog of Substance P, Cholecystokinin (CCK),        Corticotropin-releasing hormone/factor (CRH/CRF), Dermorphin,        FGF-2 or basic fibroblast growth factor, Galanin, Melanopsin,        Neurotension, cyclic RGDfK and cyclic RGDyV; preferably        bombesin, somatostatin, LHRH, EGF, VEGF, hCG, fragments of        luteinizing hormone (LH), Octreotide, Vapreotide, Lanreotide,        RC-3940 series, Decapeptyl, Lupron, Zoladex, Cetrorelix,        peptides or peptidomimetics containing the NGR or RGD motifs or        derived from these motifs such as CNGRC (linear), GNGRG        (cyclic), ACDC RGD CFCG (cyclic), CDCRGDCFC, CNGRC (cyclic),        CRGDCGG, CNGRC, or other peptides such as ATWLPPR,        Thrombospondin (TSP)-1 mimetics, (RGD peptidomimetic),        CTTHWGFTLC, CGNKRTRGC, neuropeptide substance P, SSP, the Sar9,        Met(O2)11 analog of Substance P, Cholecystokinin (CCK),        Corticotropin-releasing hormone/factor (CRH/CRF), Dermorphin,        FGF-2 or basic fibroblast growth factor, Galanin, Melanopsin,        Neurotension    -   and protein or macro-molecular targeting moieties, for example        IL-2, GM-CSF, TNF-a, transferrin, immunoglobulins,        Acetylated-LDL, Lactoferrin (Lf) (also called lactotransferrin)        and lactoferricin (Lcin), Gambogic acid (GA).

In principle, any ligand of a cell surface receptor may beadvantageously used as a targeting moiety. For instance, ATWLPPR peptideis a potent antagonist of VEGF; thrombospondin-1 (TSP-1) inducesapoptosis in endothelial cells, RGD-motif mimics block integrinreceptors, NGR-containing peptides inhibit aminopeptidase N, and cyclicpeptides containing the sequence of HWGF selectively inhibit MMP-2 andMMP-9. LyP-1 peptide specifically binds to tumor lymphatic vessels.Illustrative other ligands include peptide ligands identified fromlibrary screens, tumor cell-specific peptides, tumor cell-specificaptamers, tumor cell-specific carbohydrates, tumor cell-specificmonoclonal or polyclonal antibodies, Fab or scFv (i.e., a single chainvariable region) fragments of antibodies such as, for example, a Fabfragment of an antibody directed to EphA2 or other proteins specificallyexpressed or uniquely accessible on metastatic cancer cells, smallorganic molecules derived from combinatorial libraries, growth factors,such as EGF, FGF, insulin, and insulin-like growth factors, andhomologous polypeptides, somatostatin and its analogs, transferrin,lipoprotein complexes, bile salts, selecting, steroid hormones,Arg-Gly-Asp containing peptides, retinoids, various Galectins, δ-opioidreceptor ligands, cholecystokinin A receptor ligands, ligands specificfor angiotensin AT1 or AT2 receptors, peroxisome proliferator-activatedreceptor λ ligands, β-lactam antibiotics such as penicillin, smallorganic molecules including antimicrobial drugs, and other moleculesthat bind specifically to a receptor preferentially expressed on thesurface of tumor cells or on an infectious organism, antimicrobial andother drugs designed to fit into the binding pocket of a particularreceptor based on the crystal structure of the receptor or other cellsurface protein, ligands of tumor antigens or other moleculespreferentially expressed on the surface of tumor cells, or fragments ofany of these molecules. Examples of tumor-specific antigens that canfunction as targeting moieties include extracellular epitopes of amember of the Ephrin family of proteins, such as EphA2. EphA2 expressionis restricted to cell-cell junctions in normal cells, but EpbA2 isdistributed over the entire cell surface in metastatic tumor cells.Thus, EphA2 on metastatic cells would be accessible for binding to, forexample, a Fab fragment of an antibody conjugated to an immunogen,whereas the protein would not be accessible for binding to the Fabfragment on normal cells, resulting in a targeting moiety specific formetastatic cancer cells.

Further examples for such targeting moieties are: FSH-33, Allatostatin1, Hepatocarcinoma targeting peptide, Peptide GFE, anti-EGFR antibodiesand/or antibody fragments, in particular Cetuximab, CendR, iRGD peptide(RGD-CendR hybrid peptide), small molecules, antibodies and/or antibodyfragments binding to cancer-specific epitopes like e.g. CEA,Gastrin-releasing peptide receptors, Somatostatin receptors, Galaninreceptors, Follicle-stimulating hormone receptors, p32 protein,Fibroblast growth factor receptors, HepG2, Epidermal growth factorreceptors, Integrin αcβ6, Neuropilin-1 receptor and VEGF receptors.

The phrases “in bound form”, “connected to” or “moiety” refer tosub-structures which are part of a larger molecule. The phrases “inbound form” and “connected to” are used to simplify reference tomoieties by naming or listing reagents, starting materials orhypothetical starting materials well known in the art, and whereby “inbound form” or “connected to” mean that for example one or more hydrogenradicals (—H), or one or more activating or protecting groups present inthe reagents or starting materials are not present in the moiety whenpart of a larger molecule.

To enhance physicochemical or pharmacokinetic properties of a drug invivo, such drug can be conjugated with a carrier. If the drug, such astreprostinil, is transiently bound to a carrier and/or a linker, as inthe present invention, such systems are commonly assigned as“carrier-linked prodrugs”. According to the definitions provided byIUPAC (as given under http://www.chem.qmul.ac.uk/iupac/medchem/ah.html,accessed on Mar. 7, 2011), a carrier-linked prodrug is a prodrug thatcontains a temporary linkage of a given active substance with atransient carrier group that produces improved physicochemical orpharmacokinetic properties and that can be easily removed in vivo,usually by a hydrolytic cleavage.

The term “promoiety” refers to the part of the prodrug which is not thedrug, thus meaning linker and carrier and/or any optional spacermoieties.

The terms “reversible prodrug linkers” or “transient prodrug linkers”refer to linkers comprising, in particular consisting of reversiblelinkages that are non-enzymatically hydrolytically degradable, i.e.cleavable, under physiological conditions (aqueous buffer at pH 7.4, 37°C.) with half-lives ranging from, for example, one hour to three months.On the other hand, stable or permanent linkages are typicallynon-cleavable permanent bonds, meaning that they have a half-life of atleast six months under physiological conditions (aqueous buffer at pH7.4, 37° C.).

A “traceless prodrug linker” refers to a prodrug linker from which adrug is released in its free form, meaning that upon release from thepromoiety the drug does not contain any traces of the promoiety.

The term “polymer” describes a molecule comprising, in particularconsisting of repeating structural units connected by chemical bonds ina linear, circular, branched, crosslinked or dendrimeric way or acombination thereof, which can be of synthetic or biological origin or acombination of both. Typically, a polymer has a molecular weight of atleast 500 Da. It is understood, that when the polymer is a polypeptide,then the individual amino acids of the polypeptide may be the same ormay be different.

The term “polymeric” refers to a moiety comprising one or more polymer.

The term “poly(ethylene glycol)-based polymeric chain” or “PEG-basedpolymeric chain” refers to a polymer comprising at least 20 weight %ethylene glycol moieties, more preferably at least 50% by weight, evenmore preferably at least 80% by weight ethylene glycol moieties, whichchain is optionally capped and/or optionally further comprises one ormore functional groups, for example amine group(s). It is understoodthat a PEG-based polymeric chain may be terminated or interrupted byalkyl or aryl groups and optionally be substituted with heteroatomsand/or functional groups. Suitable capping or terminating groups for aPEG-based polymeric chain are for example CH₃—, CH₃—O— and CH₃—CH₂—.Accordingly, a PEG-based polymer is a polymer comprising at least 20weight % ethylene glycol moieties, more preferably at least 50% byweight, even more preferably at least 80% by weight ethylene glycolmoieties.

The term “hydrogel” may be defined as a three-dimensional, hydrophilicor amphiphilic polymeric network capable of taking up large quantitiesof water which causes swelling of the hydrogel in aqueous media. Thenetworks are composed of homopolymers or copolymers and are insolubledue to the presence of covalent chemical or physical (ionic, hydrophobicinteractions, entanglements) crosslinks. The crosslinks provide thenetwork structure and physical integrity.

The terms “spacer”, “spacer group”, “spacer molecule”, and “spacermoiety” are used interchangeably and refer to any moiety suitable forconnecting two moieties, such as C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl or C₂₋₅₀alkinyl, which fragment is optionally interrupted by one or more groupsselected from —NH—, —N(C₁₋₄ alkyl)-, —O—, —S—, —C(O)—, —C(O)NH—,—C(O)N(C₁₋₄ alkyl)-, —O—C(O)—, —S(O)—, —S(O)₂—, —O—C(O)NH—,—O—(CO)N(C₁₋₄ alkyl)-, 4- to 7-membered heterocyclyl, phenyl ornaphthyl. Preferably, the terms refer to C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl orC₂₋₅₀ alkinyl, which fragment is optionally interrupted by one or moregroups selected from —NH—, —N(C₁₋₄ alkyl)-, —O—, —S—, —C(O)—, —C(O)NH—,—C(O)N(C₁₋₄ alkyl)-, —O—C(O)—, —S(O)—, —S(O)₂—, 4- to 7-memberedheterocyclyl, phenyl or naphthyl.

The term “terminus” refers to the last carbon atom or heteroatom of alinear or branched chain of carbon atoms and/or heteroatoms, i.e.“terminus” refers to a carbon or heteroatom which is connected toexactly one other carbon or heteroatom.

“Terminal/terminally” or “terminally connected” means that moieties areconnected to the terminus or termini of another moiety.

“Pharmaceutical composition” or “composition” means a compositioncontaining one or more drugs or prodrugs, and optionally one or moreexcipients, as well as any product which results, directly orindirectly, from combination, complexation or aggregation of any two ormore of the excipients and/or the drug or prodrug, or from dissociationof one or more of the excipients and/or drug and/or prodrug, or fromother types of reactions or interactions of one or more of theexcipients and/or drug and/or prodrug. Accordingly, the a pharmaceuticalcomposition of the present invention encompasses any compositionobtainable by admixing a carrier-linked treprostinil prodrug of thepresent invention and a pharmaceutically acceptable excipient.

The term “excipient” refers to a diluent, adjuvant, or vehicle withwhich the carrier-linked treprostinil prodrug is administered. Suchpharmaceutical excipient can be sterile liquids, such as water and oils,including those of petroleum, animal, vegetable or synthetic origin,including but not limited to peanut oil, soybean oil, mineral oil,sesame oil and the like. Water is a preferred excipient when thepharmaceutical composition is administered orally. Saline and aqueousdextrose are preferred excipients when the pharmaceutical composition isadministered intravenously. Saline solutions and aqueous dextrose andglycerol solutions are preferably employed as liquid excipients forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, mannitol, trehalose, gelatin, malt, rice,flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc,sodium chloride, dried skim milk, glycerol, propylene, glycol, water,ethanol and the like. The composition, if desired, can also containminor amounts of wetting or emulsifying agents, pH buffering agents,like, for example, acetate, succinate, tris, carbonate, phosphate, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), MES(2-(N-morpholino)ethanesulfonic acid), or can contain detergents, likeTween, poloxamers, poloxamines, CHAPS, Igepal, or amino acids like, forexample, glycine, lysine, or histidine. These compositions can take theform of solutions, suspensions, emulsions, tablets, pills, capsules,powders, sustained-release formulations and the like. The compositioncan be formulated as a suppository, with traditional binders andexcipients such as triglycerides. Oral formulation can include standardexcipients such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Examples of suitable pharmaceutical excipients are described in“Remington's Pharmaceutical Sciences” by E. W. Martin. Such compositionswill contain a therapeutically effective amount of treprostinil in theform of at least one carrier-linked treprostinil prodrug of the presentinvention, preferably in purified form, together with a suitable amountof excipient so as to provide the form for proper administration to thepatient. The formulation should suit the mode of administration.

The term “pharmaceutically acceptable” means approved by a regulatoryagency such as the EMEA (Europe) and/or the FDA (US) and/or any othernational regulatory agency for use in animals, preferably in humans.

“Dry composition” means that the pharmaceutical composition comprisingcarrier-linked treprostinil prodrug according to the present inventionis provided in a dry form in a container. Suitable methods for dryingare spray-drying and lyophilization (freeze-drying). Such drycomposition of carrier-linked treprostinil prodrug has a residual watercontent of a maximum of 10%, preferably less than 5% and more preferablyless than 2% (determined according to Karl Fischer). The preferredmethod of drying is lyophilization. “Lyophilized composition” means thatthe pharmaceutical composition comprising carrier-linked treprostinilprodrug was first frozen and subsequently subjected to water reductionby means of reduced pressure. This terminology does not excludeadditional drying steps which may occur in the manufacturing processprior to filling the composition into the final container.

“Lyophilization” (freeze-drying) is a dehydration process, characterizedby freezing a composition and then reducing the surrounding pressureand, optionally, adding heat to allow the frozen water in thecomposition to sublime directly from the solid phase to gas. Typically,the sublimed water is collected by desublimation.

The term “functional group” refers to specific groups of atoms withinmolecules that can undergo characteristic chemical reactions. Examplesof functional groups are hydroxyl, carbonyl, aldehyde, carboxyl, ester,ketal, hemiketal, acetal, hemiacetal, primary/secondary/tertiary amine,cyanate, disulfide, sulfhydryl, sulfonyl, phosphate.

If a functional group is coupled to another functional group, theresulting chemical structure is referred to as “linkage”. For example,the reaction of an amine functional group with a carboxyl functionalgroup results in an amide linkage. Further examples for linkages areester, ether, ketal, acetal, primary/secondary/tertiary amine,carboxamide, sulfide, and disulfide.

“Alkyl” means a straight-chain (linear, unbranched) or branched carbonchain (unsubstituted alkyl). Optionally, one or more hydrogen atom(s) ofan alkyl carbon may be replaced by a substituent as indicated herein,which will be referred to as “substituted alkyl”. In general, apreferred alkyl is C₁₋₆ alkyl.

“C₁₋₄ alkyl” means an alkyl chain having 1 to 4 carbon atoms(unsubstituted C₁₋₄ alkyl), e.g. if present at the end of a molecule:methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyltert-butyl, or e.g. —CH₂—, —CH₂—CH₂—, —CH(CH₃)—, —CH₂—CH₂—CH₂—,—CH(C₂H₅)—, —C(CH₃)₂—, when two moieties of a molecule are linked by thealkyl group (also referred to as C₁₋₄ alkylene). Optionally, one or morehydrogen atom(s) of a C₁₋₄ alkyl carbon may be replaced by a substituentas indicated herein. Accordingly, “C₁₋₅₀ alkyl” means an alkyl chainhaving 1 to 50 carbon atoms.

“C₁₋₆ alkyl” means an alkyl chain having 1-6 carbon atoms, e.g. ifpresent at the end of a molecule: C₁₋₄ alkyl, methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl,or e.g. —CH₂—, —CH₂—CH₂—, —CH(CH₃)—, —C(CH₂)—, —CH₂—CH₂—CH₂—,—CH(C₂H₅)—, —C(CH₃)₂—, when two moieties of a molecule are linked by thealkyl group (also referred to as C₁₋₆ alkylene). One or more hydrogenatom(s) of a C₁₋₆ alkyl carbon may be replaced by a substituent asindicated herein. The terms C₁₋₁₅ alkyl or C₁₋₁₅ alkylene are definedaccordingly.

“C₂₋₆ alkenyl” means an alkenyl chain having 2 to 6 carbon atoms, e.g.if present at the end of a molecule: —CH═CH₂, —CH═CH—CH₃, —CH₂—CH═CH₂,—CH═CH—CH₂—CH₃, —CH═CH—CH═CH₂, or e.g. —CH═CH—, when two moieties of amolecule are linked by the alkenyl group. One or more hydrogen atom(s)of a C₂₋₆ alkenyl carbon may be replaced by a substituent as indicatedherein.

The term C₂₋₄ alkenyl is defined accordingly.

“C₂₋₆ alkynyl” means an alkynyl chain having 2 to 6 carbon atoms, e.g.if present at the end of a molecule: —C≡CH, —CH₂—C≡CH, CH₂—CH₂—C≡CH,CH₂—C≡C—CH₃, or e.g. —C≡C— when two moieties of a molecule are linked bythe alkynyl group. One or more hydrogen atom(s) of a C₂₋₆ alkynyl carbonmay be replaced by a substituent as indicated herein. The term C₂₋₄alkynyl is defined accordingly.

“C₂₋₅₀ alkenyl” means a branched or unbranched alkenyl chain having 2 to50 carbon atoms (unsubstituted C₂₋₅₀ alkenyl), e.g. if present at theend of a molecule: —CH═CH₂, —CH═CH—CH₃, —CH₂—CH═CH₂, —CH═CH—CH₂—CH₃,—CH═CH—CH═CH₂, or e.g. —CH═CH—, when two moieties of a molecule arelinked by the alkenyl group. Optionally, one or more hydrogen atom(s) ofa C₂₋₅₀ alkenyl carbon may be replaced by a substituent as furtherspecified. Accordingly, the term “alkenyl” relates to a carbon chainwith at least one carbon carbon double bond. Optionally, one or moretriple bonds may occur. The term “C₂₋₁₅ alkenyl” is defined accordingly.

“C₂₋₅₀ alkynyl” means a branched or unbranched alkynyl chain having 2 to50 carbon atoms (unsubstituted C₂₋₅₀ alkynyl), e.g. if present at theend of a molecule: —C≡CH, —CH₂—C≡CH, CH₂—CH₂—C≡CH, CH₂—C≡C—CH₃, or e.g.—C≡C— when two moieties of a molecule are linked by the alkynyl group.Optionally, one or more hydrogen atom(s) of a C₂₋₅₀ alkynyl carbon maybe replaced by a substituent as further specified. Accordingly, the term“alkynyl” relates to a carbon chain with at least one carbon triplebond. Optionally, one or more double bonds may occur. The term “C₂₋₁₅alkynyl” is used accordingly.

“C₃₋₇ cycloalkyl” or “C₃₋₇ cycloalkyl ring” means a cyclic alkyl chainhaving 3 to 7 carbon atoms, which may have carbon-carbon double bondsbeing at least partially saturated (unsubstituted C₃₋₇ cycloalkyl), e.g.cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl,cycloheptyl. Optionally, one or more hydrogen atom(s) of a cycloalkylcarbon may be replaced by a substituent as indicated herein. The term“C₃₋₇ cycloalkyl” or “C₃₋₇ cycloalkyl ring” also includes bridgedbicycles like norbonane (norbonanyl) or norbonene (norbonenyl).Accordingly, “C₃₋₅ cycloalkyl” means a cycloalkyl having 3 to 5 carbonatoms. Accordingly, “C₃₋₁₀ cycloalkyl” means a cycloalkyl having 3 to 10carbon atoms. The term “C₅₋₆ cycloalkyl” is defined accordingly.

“Halogen” means fluoro, chloro, bromo or iodo. It is generally preferredthat halogen is fluoro or chloro.

“4 to 7 membered heterocyclyl” or “4 to 7 membered heterocycle” means aring with 4, 5, 6 or 7 ring atoms that may contain up to the maximumnumber of double bonds (aromatic or non-aromatic ring which is fully,partially or un-saturated) wherein at least one ring atom up to 4 ringatoms are replaced by a heteroatom selected from the group consisting ofsulfur (including —S(O)—, —S(O)₂—), oxygen and nitrogen (including═N(O)—) and wherein the ring is linked to the rest of the molecule via acarbon or nitrogen atom (unsubstituted 4 to 7 membered heterocyclyl).For the sake of completeness it is indicated that in some embodiments ofthe present invention, 4 to 7 membered heterocyclyl has to fulfilladditional requirements. Examples for a 4 to 7 membered heterocycles areazetidine, oxetane, thietane, furan, thiophene, pyrrole, pyrroline,imidazole, imidazoline, pyrazole, pyrazoline, oxazole, oxazoline,isoxazole, isoxazoline, thiazole, thiazoline, isothiazole,isothiazoline, thiadiazole, thiadiazoline, tetrahydrofuran,tetrahydrothiophene, pyrrolidine, imidazolidine, pyrazolidine,oxazolidine, isoxazolidine, thiazolidine, isothiazolidine,thiadiazolidine, sulfolane, pyran, dihydropyran, tetrahydropyran,imidazolidine, pyridine, pyridazine, pyrazine, pyrimidine, piperazine,piperidine, morpholine, tetrazole, triazole, triazolidine,tetrazolidine, diazepane, azepine or homopiperazine. Optionally, one ormore hydrogen atom(s) of a 4 to 7 membered heterocyclyl may be replacedby a substituent. The terms “3 to 7 membered heterocyclyl” and “5 or 6membered heterocyclyl” are defined accordingly.

“8 to 11 membered heterobicyclyl” or “8 to 11 membered heterobicycle”means a heterocyclic system of two rings with 8 to 11 ring atoms, whereat least one ring atom is shared by both rings and that may contain upto the maximum number of double bonds (aromatic or non-aromatic ringwhich is fully, partially or un-saturated) wherein at least one ringatom up to 6 ring atoms are replaced by a heteroatom selected from thegroup consisting of sulfur (including —S(O)—, —S(O)₂—), oxygen andnitrogen (including ═N(O)—) and wherein the ring is linked to the restof the molecule via a carbon or nitrogen atom (unsubstituted 8 to 11membered heterobicyclyl). Examples for an 8 to 11 membered heterobicycleare indole, indoline, benzofuran, benzothiophene, benzoxazole,benzisoxazole, benzothiazole, benzisothiazole, benzimidazole,benzimidazoline, quinoline, quinazoline, dihydroquinazoline, quinoline,dihydroquinoline, tetrahydroquinoline, decahydroquinoline, isoquinoline,decahydroisoquinoline, tetrahydroisoquinoline, dihydroisoquinoline,benzazepine, purine or pteridine. The term 8 to 11 memberedheterobicycle also includes spiro structures of two rings like1,4-dioxa-8-azaspiro[4.5]decane or bridged heterocycles like8-aza-bicyclo[3.2.1]octane. The term “9 to 11 membered heterobicyclyl”or “9 to 11 membered heterobicycle” is defined accordingly.

The term “aliphatic” means fully saturated.

The term “interrupted” means that between two carbon atoms of, forexample, a linker or a spacer or at the respective end of the carbonchain between the respective carbon atom and the hydrogen atom a group(such a —O— or —NH—) is inserted.

In general the term “substituted” preferably refers to substituents,which are the same or different and which are independently selectedfrom the group consisting of halogen, CN, COOR^(b9), OR^(b9),C(O)R^(b9), C(O)N(R^(b9)R^(b9a)), S(O)₂N(R^(b9)R^(b9a)),S(O)N(R^(b9)R^(b9a)), S(O)₂R^(b9), S(O)R^(b9),N(R^(b9))S(O)₂N(R^(b9a)R^(b9b)), SR^(b9), N(R^(b9)R^(b9a)), NO₂,OC(O)R^(b9), N(R^(b9))C(O)R^(b9a), N(R^(b9))S(O)₂R^(b9a),N(R^(b9))S(O)R^(b9a), N(R^(b9))C(O)OR^(b9a),N(R^(b9))C(O)N(R^(b9a)R^(b9b)), OC(O)N(R^(b9)R^(b9a)), T^(b), C₁₋₅₀alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl,

-   -   wherein T^(b), C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are        optionally substituted with one or more R^(b10), which are the        same or different, and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and        C₂₋₅₀ alkynyl are optionally interrupted by one or more groups        selected from the group consisting of T^(b), —C(O)O—; —O—;        —C(O)—; —C(O)N(R^(b11))—; —S(O)₂N(R^(b11))—; —S(O)N(R^(b11))—;        —S(O)₂—; —S(O)—; —N(R^(b11))S(O)₂N(R^(b11a))—; —S—;        —N(R^(b11))—; —OC(O)R^(b11); —N(R^(b11))C(O)—; —N(R^(b11))S(O)₂;        —N(R^(b11))S(O)—; —N(R^(b11))C(O)O—;        —N(R^(b11))C(O)N(R^(b11a))—; and —OC(O)N(R^(b11)R^(b11a));    -   R^(b9), R^(b9a), R^(b9b) are independently selected from the        group consisting of H; T^(b); and C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl;        and C₂₋₅₀ alkynyl,        -   wherein T^(b), C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl            are optionally substituted with one or more R^(b10), which            are the same or different, and wherein C₁₋₅₀ alkyl; C₂₋₅₀            alkenyl; and C₂₋₅₀ alkynyl are optionally interrupted by one            or more groups selected from the group consisting of T^(b),            —C(O)O—, —O—, —C(O)—, —C(O)N(R^(b))—, —S(O)₂N(R^(b11))—,            —S(O)N(R^(b11))—, —S(O)₂—, —S(O)—,            —N(R^(b11))S(O)₂N(R^(b11a))—, —S—, —N(R^(b11))—,            —OC(O)R^(b11), —N(R^(b11))C(O)—, —N(R^(b11))S(O)₂—,            —N(R^(b11))S(O)—, —N(R^(b11))C(O)O—,            —N(R^(b11))C(O)N(R^(b11a))—, and —OC(O)N(R^(b11)R^(b11a)),        -   T^(b) is selected from the group consisting of phenyl,            naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 4-            to 7-membered heterocyclyl, and 9- to 11-membered            heterobicyclyl, wherein T^(b) is optionally substituted with            one or more R^(b10), which are the same or different,        -   R^(b10) is halogen, CN, oxo (═O), COOR^(b12), OR^(b12),            C(O)R^(b12), C(O)N(R^(b12)R^(b12a)),            S(O)₂N(R^(b12)R^(b12a)), S(O)N(R^(b12)R^(b12a)),            S(O)₂R^(b12), S(O)R^(b12),            N(R^(b12))S(O)₂N(R^(b12a)R^(b12b)), SR^(b12),            N(R^(b12)R^(b12a)), NO₂, OC(O)R^(b12),            N(R^(b12))C(O)R^(b12a), N(R^(b12))S(O)₂R^(b12a),            N(R^(b12))S(O)R^(b12a), N(R^(b12))C(O)OR^(b12a),            N(R^(b12))C(O)N(R^(b12a)R^(b2b)),        -   OC(O)N(R^(b12)R^(b12a)), or C₁₋₆ alkyl, wherein C₁₋₆ alkyl            is optionally substituted with one or more halogen, which            are the same or different,        -   R^(b11), R^(b1a), R^(b12), R^(b12a), R^(b12b) are            independently selected from the group consisting of H; or            C₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionally substituted            with one or more halogen, which are the same or different.

The term “interrupted” means that between two carbons a group isinserted or that at the end of the carbon chain between the carbon andhydrogen.

Generally, the term “dashed line” which is used to indicate theconnection of one moiety to another is different from a dashed bondwhich is used to indicate stereochemistry. The person skilled in the artwill be able to distinguish between these two.

In general the term “comprise” or “comprising” also encompasses “consistof” or “consisting of”.

The term “water soluble” as in a “water-soluble carrier” is a carrierthat is soluble in water at room temperature. Typically, a solution of awater-soluble carrier will transmit at least about 75%, more preferablyat least about 95% of light, transmitted by the same solution afterfiltering. On a weight basis, a water-soluble carrier or parts thereofwill preferably be at least about 35% (by weight) soluble in water, morepreferably at least about 50% (by weight) soluble in water, still morepreferably about 70% (by weight) soluble in water, and still morepreferably about 85% (by weight) soluble in water. It is most preferred,however, that the water-soluble carrier or parts thereof is about 95%(by weight) soluble in water or completely soluble in water.

The carrier-linked treprostinil prodrugs according to the presentinvention comprise treprostinil moieties. Treprostinil as such is a drugknown to a person skilled in the art either in its pure form or as apharmaceutically acceptable salt thereof.

As used herein a single treprostinil compound dose is given in mg andconcentration of a treprostinil compound in a pharmaceutical compositionis given in mg/mL. As the treprostinil compound is a carrier-linkedprodrug, the concentration is based on quantitative release of freetreprostinil from the prodrug. By methods well-known in the art,aliquots of a composition are subjected to treprostinil-releasingconditions (aqueous buffer pH 7.4, 37° C., or accelerated conditions atelevated pH), until no significant increase in treprostinilconcentration is observed and the total amount of released treprostinilis determined.

In the present invention, the carrier-linked treprostinil prodrug or apharmaceutically acceptable salt thereof does not contain treprostinilin its free form or as a pharmaceutically acceptable salt thereof, butin bound form. Treprostinil is bound via one of its functional groups,e.g. via a hydroxyl or carboxyl, to the rest of the molecule and is aspart of a moiety T which is connected to a moiety X⁰ or—if m1 and m2 areboth 0—to a moiety Z¹ of formula (I). This means that the carrier-linkedtreprostinil prodrug according to the present invention containstreprostinil as a biologically active moiety. Due to the cleavage of thebiologically active moiety from the carrier-linked treprostinil prodrugwhen administered to a patient in need thereof, treprostinil is releasedeither in its free form or as a pharmaceutically acceptable saltthereof. In other words, the carrier-linked treprostinil prodrugcontains one or more moieties T, which moiety T is each substituted witha moiety X⁰ (provided that at least one of m1 and m2 is 1), which inturn is covalently bound to a carrier Z¹. Said carrier comprises acovalently bound polymer, preferably a pharmaceutically acceptablepolymer with a molecular weight of at least 500 Dalton.

In another preferred embodiment, the molecular weight of the polymer,preferably a pharmaceutically acceptable polymer is up to 160 kDa,preferably up to about 100 kDa, even more preferably up to about 50 kDa.

Preferably, a moiety X⁰ (provided that at least one of m1 and m2 is 1)and a moiety T are connected through a carbonate or ester linkage, mostpreferably a moiety X⁰ and a moiety T are connected through an esterlinkage.

Preferably, a moiety X⁰ is unsubstituted. More preferably, each moietyX⁰ is unsubstituted.

In one preferred embodiment, m1 is 0 and m2 is 1.

In another preferred embodiment, both m1 and m2 are 0.

In another preferred embodiment, both m1 and m2 are 1.

Preferably, a sub-structure X⁰—Z¹ is C(R¹R²)—CH₂—Z¹, wherein R¹, R² areindependently selected from the group consisting of H and C₁₋₄ alkyl,provided that at least one of R¹, R² is other than H; or (CH₂)_(n)—Z¹,wherein n is 2, 3, 4, 5, 6, 7 or 8.

Preferably, the carrier Z¹ is covalently attached to a moiety X⁰ via anamide group.

Preferably, R³ is halogen; CN; C(O)R⁴; C(O)OR⁴; OR⁴; C(O)R⁴;C(O)N(R⁴R^(4a)); S(O)₂N(R⁴R^(4a)); S(O)N(R⁴R^(4a)); S(O)₂R⁴; S(O)R⁴;N(R⁴)S(O)₂N(R^(4a)R^(4b)); SR⁴; N(R⁴R^(4a)); NO₂; OC(O)R⁴;N(R⁴)C(O)R^(4a); N(R⁴)SO₂R^(4a); N(R⁴)S(O)R^(4a);N(R⁴)C(O)N(R^(4a)R^(4b)) N(R⁴)C(O)OR^(4a); OC(O)N(R⁴R^(4a)); or T⁰.

Preferably, R⁴, R^(4a), R^(4b) are independently selected from the groupconsisting of H; T⁰; C₁₋₄ alkyl; C₂₋₄ alkenyl; and C₂₋₄ alkynyl, whereinC₁₋₄ alkyl; C₂₋₄ alkenyl; and C₂₋₄ alkynyl are optionally substitutedwith one or more R⁵, which are the same of different.

More preferably, the carrier-linked treprostinil prodrug is of formula(II):

-   -   wherein each T is independently selected from structures (i)        to (v) (preferably (iii)):

-   -   wherein dashed lines indicating attachment to the rest of the        molecule;    -   y is an integer ranging of from 1 to 64, preferably ranging from        1 to 16, more preferably y is selected from 1, 2, 3, 4, 5, 6, 7        8, 9, 10, 11, 12, 13, 14, 15 and 16, even more preferably y is        4, 6, 8, 10 or 12; even more preferably, y is 4, 6, or 8; most        preferably 4;    -   R^(a1) is selected from the group of unsubstituted alkyl;        substituted alkyl; unsubstituted phenyl; substituted phenyl;        unsubstituted naphthyl; substituted naphthyl; unsubstituted        indenyl; substituted indenyl; unsubstituted indanyl; substituted        indanyl; unsubstituted tetralinyl; substituted tetralinyl;        unsubstituted C₃₋₁₀ cycloalkyl; substituted C₃₋₁₀ cycloalkyl;        unsubstituted 4- to 7-membered heterocyclyl; substituted 4- to        7-membered heterocyclyl; unsubstituted 9- to 11-membered        heterobicyclyl; and substituted 9- to 11-membered        heterobicyclyl;    -   R^(a2) is selected from H, unsubstituted alkyl, and substituted        alkyl;    -   R^(a3) and R^(a4) are independently selected from the group        consisting of H, unsubstituted alkyl, and substituted alkyl;    -   n is 0 or 1;    -   optionally, R^(a1) and R^(a3) are joined together with the atoms        to which they are attached to form a ring A;    -   A is selected from phenyl; naphthyl; indenyl; indanyl;        tetralinyl; C₃₋₁₀ cycloalkyl; 4- to 7-membered aliphatic        heterocyclyl; or 9- to 11-membered aliphatic heterobicyclyl,        wherein A is unsubstituted or substituted;    -   preferably, A is selected from the group consisting of C₃₋₁₀        cycloalkyl; 4- to 7-membered aliphatic heterocyclyl; and 9- to        11-membered aliphatic heterobicyclyl, wherein A is unsubstituted        or substituted;    -   Q is a spacer moiety;    -   Z¹ is a carrier comprising a covalently bound polymer,        preferably a pharmaceutically acceptable polymer;    -   or a pharmaceutical salt thereof.

Preferably, R^(a1) is C₁₋₆ alkyl or substituted C₁₋₆ alkyl, morepreferably C₁₋₄ alkyl or substituted C₁₋₄ alkyl.

More preferably, R^(a1) is selected from methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, and benzyl.

Preferably, R^(a2) is H.

Preferably, R^(a3) is H, C₁₋₆ alkyl or substituted C₁₋₆ alkyl, morepreferably C₁₋₄ alkyl or substituted C₁₋₄ alkyl. More preferably, R^(a3)is selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, t-butyl, and benzyl.

More preferably, R^(a3) is H.

Preferably, R^(a4) is selected from H, C₁₋₆ alkyl or substituted C₁₋₆alkyl, more preferably C₁₋₄ alkyl or substituted C₁₋₄ alkyl. Morepreferably, R^(a4) is selected from methyl, ethyl, n-propyl, isopropyl,n-butyl, isobutyl, sec-butyl, t-butyl, and benzyl.

More preferably, R^(a4) is H.

In another preferred embodiment, R^(a1) and R^(a3) are joined togetherwith the atoms to which they are attached to form a ring A; wherein A isselected from the group consisting of cyclopropane, cyclobutane,cyclopentane, cyclohexane, cycloheptane. Even more preferred iscyclohexane.

Preferably, Q in formula (II) is selected from COOR^(a9); OR^(a9);C(O)R^(a9); C(O)N(R^(a9)R^(a9a)); S(O)₂N(R^(a9)R^(a9a));S(O)N(R^(a9)R^(a9a)); S(O)₂R^(a9); S(O)R^(a9);N(R^(a9))S(O)₂N(R^(a9a)R^(a9b)); SR^(a9); N(R^(a9)R^(a9a)); OC(O)R^(a9);N(R⁹)C(O)R^(a9a); N(R^(a9)))S(O)₂R^(a9a); N(R^(a9))S(O)R^(a9a);N(R⁹)C(O)OR^(a9a); N(R^(a9))C(O)N(R^(a9a)R^(a9b));OC(O)N(R^(a9)R^(a9a)); W; C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl,wherein W, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionallysubstituted with one or more R^(a10), which are the same or different,

and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionallyinterrupted by one or more groups selected from the group consisting of—W—, —C(O)O—; —O—; —C(O)—; —C(O)N(R^(a11))—; —S(O)₂N(R^(a11))—;—S(O)N(R^(a9))—; —S(O)₂—; —S(O)—; —N(R^(a11))S(O)₂N(R^(a11a))—; —S—;—N(R^(a11))—; —OC(O)R^(a11); —N(R^(a11))C(O)—; —N(R^(a11))S(O);—N(R^(a11))S(O)—; —N(R^(a11))C(O)O—; —N(R^(a11))C(O)N(R^(a11a))—; and—OC(O)N(R^(a11)R^(a11a));

R^(a9), R^(a9a), R^(a9b) are independently selected from the groupconsisting of H; W; and C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl,

wherein W, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionallysubstituted with one or more R^(a10), which are the same or different,

and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionallyinterrupted by one or more groups selected from the group consisting ofW, —C(O)O—; —O—; —C(O)—; —C(O)N(R^(a11))—; —S(O)₂N(R^(a11))—;—S(O)N(R^(a11))—; —S(O)₂—; —S(O)—; —N(R^(a11))S(O)₂N(R^(a11a))—; —S—;—N(R^(a11))—; —OC(O)R^(a11); —N(R^(a11))C(O)—; —N(R^(a11))S(O)₂—;—N(R^(a11))S(O)—; —N(R^(a11))C(O)O—; —N(R^(a11))C(O)N(R^(a11a))—; and—OC(O)N(R^(a11)R^(a11a));

W is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4- to 7-membered heterocyclyl; or9- to 11-membered heterobicyclyl, wherein W is optionally substitutedwith one or more R^(a10), which are the same or different;

R^(a10) is halogen; CN; oxo (═O); COOR^(a12); OR^(a12); C(O)R^(a12);C(O)N(R^(a12)R^(a12a)); S(O)₂N(R^(a12)R^(a12a)); S(O)N(R^(a12)R^(a12a));S(O)₂R^(a12); S(O)R^(a12); N(R^(a12))S(O)₂N(R^(a12a)R^(a12b)); SR^(a12);N(R^(a12)R^(a12a)); NO₂; OC(O)R^(a12); N(R^(a12))C(O)R^(a12a);N(R^(a12))S(O)₂R^(a12a); N(R^(a12))S(O)R^(a2a); N(R^(a12))C(O)OR^(a12a);N(R^(a12))C(O)N(R^(a12a)R^(a12b)); OC(O)N(R^(a12)R^(a12a)); or C₁₋₆alkyl, wherein C₁₋₆ alkyl is optionally substituted with one or morehalogen, which are the same or different;

R^(a11), R^(a11a), R^(a12), R^(a2a), R^(a12b) are independently selectedfrom the group consisting of H; and C₁₋₆ alkyl, wherein C₁₋₆ alkyl isoptionally substituted with one or more halogen, which are the same ordifferent.

More preferably, each -Q- is independently -Q^(1a)Q¹-*, wherein theasterisk indicates the connection to Z¹ and wherein

Q^(1a) is a bond; —C(O)O—; —O—; —C(O)—; —C(O)N(R^(a9a))—;—S(O)₂N(R^(a9a))—; —S(O)N(R^(a9a))—; —S(O)—; —S(O)—;—N(R^(a9a))S(O)₂N(R^(a9b))—; —S—; —N(R^(a9a))—; —OC(O)—;—N(R^(a9a))C(O)—; —N(R^(a9a))S(O)₂—; —N(R^(a9a))S(O)—;—N(R^(a9a))C(O)O—; —N(R^(a9a))C(O)N(R^(a9b))—; —OC(O)N(R^(a9a))—; or—W—, preferably —C(O)N(R^(a9a))— or —N(R^(a9a))C(O)—;

Q¹ is selected from C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl whichare optionally substituted with one or more R^(a10), which areoptionally interrupted, provided that Q¹ is at least C₂, by one or moregroups selected from the group consisting of C₃₋₇ cycloalkyl, 4- to7-membered heterocyclyl,

wherein each of said group may individually be present one or moretimes; and which C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl mayoptionally be terminated at the end connected to Z¹ by a group selectedfrom C₃₋₇ cycloalkyl, 4- to 7-membered heterocyclyl,

R^(a10) is halogen; CN; oxo (═O); COOR^(a12); OR^(a12); C(O)R^(a12);C(O)N(R^(a12)R^(a12a)); S(O)₂N(R^(a12)R^(a12a)); S(O)N(R^(a12)R^(a12a));S(O)₂R^(a12); S(O)R^(a12); N(R^(a12))S(O)₂N(R^(a12a)R^(a12b)); SR^(a12);N(R^(a12)R^(a12a)); NO₂; OC(O)R^(a12); N(R^(a12))C(O)R^(a12a);N(R^(a12))S(O)₂R^(a12a); N(R^(a12))S(O)R^(a12a);N(R^(a12))C(O)OR^(a12a); N(R^(a12))C(O)N(R^(a12a)R^(a12b));OC(O)N(R^(a12)R^(a12a)); or C₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different;

R^(a12), R^(a12a) and R^(a12b) are independently selected from the groupconsisting of H; and C₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different,

R¹³ and R^(13a) are independently selected from H, C₁₋₆ alkyl, C₂₋₆alkenyl, or C₂, alkynyl; preferably, R¹³ and R^(13a) are independentlyselected from C₁₋₆ alkyl, C₂₋₆ alkenyl, or C₂, alkynyl.

In formula (I) the moiety X⁰ is of formula (IIa):

-   -   wherein    -   the dashed line marked with the asterisk indicates attachment to        T and the unmarked dashed line indicates attachment to the rest        of the carrier-linked treprostinil prodrug; and    -   wherein Q, R^(a1), R^(a2), R^(a3), and R^(a4) are used as        defined in formula (II).

Even more preferably, the carrier-linked treprostinil prodrug has thestructure of formula (II-A):

-   -   wherein    -   each T is independently selected from structures (i) to (v):

-   -   wherein dashed lines indicating attachment to the rest of the        molecule;    -   y is an integer ranging of from 1 to 64, preferably ranging from        1 to 16, more preferably y is selected from 1, 2, 3, 4, 5, 6, 7        8, 9, 10, 11, 12, 13, 14, 15 and 16, even more preferably y is        4, 6, 8, 10 or 12; most preferably 4;    -   R^(a2) is selected from H, unsubstituted alkyl, and substituted        alkyl;    -   R^(a4) is selected from the group consisting of H, unsubstituted        alkyl, and substituted alkyl;    -   A is selected from phenyl; naphthyl; indenyl; indanyl;        tetralinyl; C₃₋₁₀ cycloalkyl; 4- to 7-membered aliphatic        heterocyclyl; or 9- to 11-membered aliphatic heterobicyclyl,        wherein A is unsubstituted or substituted;    -   Q is a spacer moiety;    -   Z¹ is a carrier comprising a covalently bound polymer,        preferably a pharmaceutically acceptable polymer.

Preferably, R^(a2) of formula (I-A) is H.

Preferably, R^(a4) of formula (I-A) is selected from H, C₁₋₆ alkyl orsubstituted C₁₋₆ alkyl, more preferably C₁₋₄ alkyl or substituted C₁₋₄alkyl. More preferably, R^(a4) is selected from methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, t-butyl, and benzyl.

More preferably, R^(a4) of formula (I-A) is H.

Preferably, Q in formula (II-A) is selected from COOR^(a9); OR⁹;C(O)R^(a9); C(O)N(R^(a9)R^(a9a)); S(O)₂N(R^(a9)R^(a9a));S(O)N(R^(a9)R^(a9a)); S(O)₂R^(a9); S(O)R^(a9);N(R^(a9))S(O)₂N(R^(a9a)R^(a9b)); SR^(a9); N(R^(a9)R^(a9a)); OC(O)R^(a9);N(R^(a9))C(O)R^(a9a); N(R^(a9))S(O)₂R^(a9a); N(R^(a9))S(O)R^(a9a);N(R^(a9))C(O)OR^(a9a); N(R^(a9))C(O)N(R^(a9a)R^(a9b));OC(O)N(R^(a9)R^(a9a)); W; C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl,wherein W, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionallysubstituted with one or more R^(a10), which are the same or different,

and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionallyinterrupted by one or more groups selected from the group consisting of—W—, —C(O)O—; —O—; —C(O)—; —C(O)N(R^(a11))—; —S(O)₂N(R^(a11))—;—S(O)N(R^(a11))—; —S(O)₂—; —S(O)—; —N(R^(a11))S(O)₂N(R^(a11a))—; —S—;—N(R^(a11))—; —OC(O)R^(a11); —N(R^(a11))C(O)—; —N(R^(a11))S(O)₂—;—N(R^(a11))S(O)—; —N(R^(a11))C(O)O—; —N(R^(a11))C(O)N(R^(a11a))—; and—OC(O)N(R^(a11)R^(a11a));

R^(a9), R^(a9a), R^(a9b) are independently selected from the groupconsisting of H; W; and C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl,

wherein W, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionallysubstituted with one or more R^(a10), which are the same or different,

and wherein C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionallyinterrupted by one or more groups selected from the group consisting ofW, —C(O)O—; —O—; —C(O)—; —C(O)N(R^(a11))—; —S(O)₂N(R^(a11))—;—S(O)N(R^(a9))—; —S(O)₂—; —S(O)—; —N(R^(a11))S(O)₂N(R^(a11a))—; —S—;—N(R^(a11))—; —OC(O)R^(a11); —N(R^(a11))C(O)—; —N(R^(a11))S(O)₂—;—N(R^(a11))S(O)—; —N(R^(a11))C(O)O—; —N(R^(a11))C(O)N(R^(a11a))—; and—OC(O)N(R^(a11)R^(a11a));

W is selected from the group consisting of phenyl; naphthyl; indenyl;indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4- to 7-membered heterocyclyl; or9- to 11-membered heterobicyclyl, wherein W is optionally substitutedwith one or more R^(a10), which are the same or different;

R^(a10) is halogen; CN; oxo (═O); COOR^(a12); OR^(a12); C(O)R^(a12);C(O)N(R^(a12)R^(a12a)); S(O)₂N(R^(a12)R^(a12a)); S(O)N(R^(a12)R^(a12a));S(O)₂R^(a12); S(O)R^(a12); N(R^(a12))S(O)₂N(R^(a12a)R^(a12b)); SR^(a12);N(R^(a12)R^(a12a)); NO₂; OC(O)R^(a12); N(R^(a12))C(O)R^(a12a);N(R^(a12))S(O)₂R^(a12a); N(R^(a12))S(O)R^(a12a);N(R^(a12))C(O)OR^(a12a); N(R^(a12))C(O)N(R^(a12a)R^(a12b));OC(O)N(R^(a12)R^(a12a)); or C₁₋₆ alkyl, wherein C₁₋₆ alkyl is optionallysubstituted with one or more halogen, which are the same or different;

R^(a11), R^(a11a), R^(a12), R^(a12a), R^(a12b) are independentlyselected from the group consisting of H; and C₁₋₆ alkyl, wherein C₁₋₆alkyl is optionally substituted with one or more halogen, which are thesame or different.

More preferably, the carrier-linked treprostinil prodrug is of formula(IIaa):

-   -   wherein    -   each T is independently selected from structures (i) or (iii):

-   -   wherein dashed lines indicate attachment to the rest of the        molecule;    -   y is an integer ranging of from 1 to 64, preferably ranging from        1 to 16, more preferably y is selected from 1, 2, 3, 4, 5, 6, 7        8, 9, 10, 11, 12, 13, 14, 15 and 16, even more preferably y is        4, 6, 8, 10 or 12; most preferably 4;    -   R^(a2) is selected from H, unsubstituted alkyl, and substituted        alkyl; preferably, R^(a2) is selected from H and substituted or        unsubstituted C₁₋₆ alkyl;    -   R^(a4) is selected from the group consisting of H, unsubstituted        alkyl, and substituted alkyl; preferably, R^(a4) is selected        from H and substituted or unsubstituted C₁₋₆ alkyl;    -   ring A¹ is a C₃₋₁₀ cycloalkyl; 4- to 7-membered aliphatic        heterocyclyl; or 9- to 11-membered aliphatic heterobicyclyl,        wherein A¹ is unsubstituted or substituted;    -   Q¹ is selected from C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀        alkynyl which are optionally substituted with one or more        R^(a10), which are optionally interrupted, provided that Q¹ is        at least C₂, by one or more groups selected from the group        consisting of C₃₋₇ cycloalkyl, 4- to 7-membered heterocyclyl,

-   -   wherein each of said group may individually be present one or        more times; and which C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀        alkynyl may optionally be terminated at the end connected to Z¹        by a group selected from C₃₋₇ cycloalkyl, 4- to 7-membered        heterocyclyl,

-   -   R^(a10) is halogen; CN; oxo (═O); COOR^(a12); OR^(a12);        C(O)R^(a12); C(O)N(R^(a12)R^(a2a)); S(O)₂N(R^(a12)R^(a12a));        S(O)N(R^(a12)R^(a12a)); S(O)₂R^(a12); S(O)R^(a12);        N(R^(a12))S(O)₂N(R^(a12a)R^(a12b)); SR^(a12);        N(R^(a12)R^(a12a)); NO₂; OC(O)R^(a12); N(R^(a12))C(O)R^(a12a);        N(R^(a12))S(O)₂R^(a12a); N(R^(a12))S(O)R^(a12a);        N(R^(a12))C(O)OR^(a12a); N(R^(a12))C(O)N(R^(a12a)R^(a12b));        OC(O)N(R^(a12)R^(a12a)); or C₁₋₆ alkyl, wherein C₁₋₆ alkyl is        optionally substituted with one or more halogen, which are the        same or different;    -   R^(a12), R^(a12a) and R^(a12b) are independently selected from        the group consisting of H; and C₁₋₆ alkyl, wherein C₁₋₆ alkyl is        optionally substituted with one or more halogen, which are the        same or different,    -   R¹³ and R^(13a) are independently selected from H, C₁₋₆ alkyl,        C₂₋₆ alkenyl, or C₂₋₆ alkynyl; preferably, R¹³ and R^(13a) are        independently selected from C₁₋₆ alkyl, C₂₋₆ alkenyl, or C₂₋₆        alkynyl;    -   Z¹ is a carrier comprising a covalently bound polymer,        preferably a pharmaceutically acceptable polymer.

It is understood that R^(a4) and R^(a2) of formula (IIaa) are vicinal.

Preferably, the carrier-linked treprostinil prodrug is of formula(IIab):

-   -   wherein    -   T, R^(a2), R^(a4), A¹, Q¹, Z¹ and y are used as defined in        formula (IIaa).

It is understood that R^(a4) and R^(a2) of formula (IIab) are vicinal.

Preferably, A¹ of formula (IIaa) or (IIab) is selected fromcyclopentane, cyclohexane or cycloheptane. More preferably, A¹ iscyclohexan.

Preferably, R^(a2) and R^(a4) of formula (IIaa) or (IIab) are both H.

Preferably, Q¹ of formula (IIaa) or (IIab) is selected from C₁₋₅₀ alkyl,which is optionally substituted with one or more R^(a10), which are thesame or different; and wherein the C₁₋₅₀ alkyl is optionallyinterrupted, provided that Q¹ is at least C₂, by one or more groupsselected from the group consisting of C₃, cycloalkyl, 4 to 7 memberedheterocyclyl,

-   -   wherein each of said group may individually be present one or        more times and which C₁₋₅₀ alkyl may optionally be terminated at        the end connected to Z¹ by a group selected from C₃₋₇        cycloalkyl, 4- to 7-membered heterocyclyl,

-   -   and wherein R¹³ and R^(13a) are used as defined for formula        (IIaa);

Preferably, y of formula (IIaa) or (IIab) is 4, 6 or 8. More preferablyy of formula (IIaa) or (IIab) is 4 or 8, most preferably y of formula(IIaa) or (IIab) is 4.

Even more preferably, the carrier-linked treprostinil prodrug is offormula (IIac) or (IIad):

-   -   wherein    -   T, Z¹ and y are used as defined in formula (IIaa),    -   x is selected from 2, 3, 4, 5, 6, 7 or 8, more preferably, x is        selected from 3, 4, 5, 6, 7, or 8,    -   X₁ is selected from C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀        alkynyl which are optionally substituted with one or more        R^(a10), and which C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl        are optionally interrupted, provided that X₁ is at least C₂, by        one or more groups selected from the group consisting of C₃₋₇        cycloalkyl, 4- to 7-membered heterocyclyl,

-   -   wherein each of said groups may individually be present one or        more times; and which C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀        alkynyl may optionally be terminated at the end connected to Z¹        by a group selected from C₃₋₇ cycloalkyl, 4- to 7-membered        heterocyclyl,

-   -   R^(a10) is halogen; CN; oxo (═O); COOR^(a12); OR^(a12);        C(O)R^(a12); C(O)N(R^(a12)R^(a12a)); S(O)₂N(R^(a12)R^(a12a));        S(O)N(R^(a12)R^(a12a)); S(O)₂R^(a12); S(O)R^(a12);        N(R^(a12))S(O)₂N(R^(a12a)R^(a12b)); SR^(a12);        N(R^(a12)R^(a12a)); NO₂; OC(O)R^(a12); N(R^(a12))C(O)R^(a12a);        N(R^(a12))S(O)₂R^(a12a); N(R^(a12))S(O)R^(a12a);        N(R^(a12))C(O)OR^(a12a); N(R^(a12))C(O)N(R^(a12a)R^(a12b));        OC(O)N(R^(a12)R^(a12a)); or C₁₋₆ alkyl, wherein C₁₋₆ alkyl is        optionally substituted with one or more halogen, which are the        same or different;    -   R^(a12), R^(a12a) and R^(a12b) are independently selected from        the group consisting of H; and C₁₋₆ alkyl, wherein C₁₋₆ alkyl is        optionally substituted with one or more halogen, which are the        same or different,    -   and wherein R¹³ and R^(13a) are used as defined for formula        (IIaa).

Preferably, X₁ of formula (IIac) and (IIad) is selected from C₁₋₁₅alkyl, C₂₋₁₅ alkenyl and C₂₋₁₅ alkynyl, which are optionally substitutedor interrupted by one or more groups selected from the group consistingof C₃₋₇ cycloalkyl, 4- to 7-membered heterocyclyl,

-   -   wherein    -   R¹³ and R^(13a) are independently selected from H, C₁₋₆ alkyl,        C₂₋₆ alkenyl, or C₂₋₆ alkynyl.

More preferably, y of formulas (IIac) or (IIad) is 4, 6, or 8, even morepreferabably y is 4 or 8, most preferably y is 4.

More preferably, x of formulas (IIac) or (IIad) is 4, 5, or 6, mostpreferably x is 6.

Even more preferably, the carrier-linked treprostinil prodrug is offormula (IIb):

wherein

Z¹ is a carrier comprising a covalently bound polymer, preferably apharmaceutically acceptable polymer,

X₁ and y are used as defined in formula (IIac) and (IIad), and

x is selected from 2, 3, 4, 5, 6, 7 or 8.

More preferably, y of formula (IIb) is 4, 6, or 8, even morepreferabably y is 4 or 8, and most preferably y is 4.

More preferably, x of formula (IIb) is 4, 5, or 6, even more preferablyx is 5 or 6, and most preferably x is 6.

Most preferably, y in formula (IIb) is 4 and x in formula (IIb) is 6.

More preferably, the carrier-linked treprostinil prodrug of formula(IIb) has the structure of formula (IIba):

wherein Z¹ is used as defined in formula (IIaa) and y is used as definedin formula (IIac) and (IIad).

Preferably, y of formula (IIba) is 4, 6 or 8. More preferably y offormula (IIba) is 4 or 8, most preferably y of formula (IIba) is 4.

More preferably, X₁ of formula (IIac) is

wherein q is selected from 1 to 4; preferably, q is 1.

More preferably, X¹ of formula (IIad) is

wherein q is selected from 1, 2, 3, or 4 and preferably is 2.

Preferably, X⁰ of formula (I) is selected from the following structures:

-   -   wherein dashed lines marked with an asterisk indicate attachment        to T and    -   unmarked dashed lines indicate attachment to the rest of the        carrier-linked treprostinil prodrug.

Preferably, all moieties T of the carrier-linked treprostinil prodrug offormula (I), (II), (IIaa), (IIab), (IIac) and (IIad) have the samestructure.

Preferably, all moieties T of formula (I) have the structure of formula(v) or formula (ii) or formula (iii), more preferably the structure offormula (v).

Preferably, all moieties T of formula (II), (IIaa), (IIab), (IIac) and(IIad) have the same structure and are either of formula (ii), (iii) or(iv). More preferably, all moieties T of formula (II), (IIaa), (IIab),(IIac) and (IIad) have the same structure and are either of formula (ii)or (iv).

Preferred sub-structures —X⁰—T of formula (I) are selected from thefollowing structures:

-   -   wherein the dashed lines indicate attachment to Z¹.

In another preferred embodiment the carrier-linked treprostinil prodrughas the structure of formula (IId):

-   -   wherein the dashed line indicates attachment to T of formula        (I);    -   Y^(v) is —N(R^(1v))—;    -   X^(v) is —C(R^(4v))(R^(4av))—; —N(R^(4v))—; —O—;        —C(R^(4v))(R^(4av))—C(R^(5v))(R^(5av))—;        —C(R^(4v))(R^(4av))—N(R^(6v))—; —N(R^(6v))—C(R^(4v))(R^(4av))—;        —C(R^(4v))(R^(4av))—O—; —O—C(R^(4v))(R^(4av))—;        —C(O)—N(R^(6v))—; or —N(R^(6v))—C(O)—;    -   X^(1v) is

-   -   X^(2v) is —C(R^(7v))(R^(7av))—; or        —C(R^(7v))(R^(7av))—C(R^(8v))(R^(8av))—;    -   X^(3v) is ═O; ═S; or ═N—CN;    -   R^(1v), R^(1av), R^(2v), R^(2av), R^(3v), R^(3av), R^(4v),        R^(4av), R^(5av), R^(6v), R^(7v), R^(7av), R^(8v), R^(8av) are        independently selected from the group consisting of H; C₁₋₆        alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, C₁₋₂₀ heteroalkyl and        Y₁-T^(v); and independently none, one or more of the pairs        R^(1av)/R^(4av), R^(1av)/R^(5av), R^(4av)/R^(5av),        R^(7av)/R^(8av) are absent and the corresponding carbon atoms to        which they are attached form a cis double bond;    -   Y^(1v) is a chemical bond or C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆        alkynyl;    -   T^(v) is selected from the group consisting of phenyl; naphthyl;        indenyl; indanyl; tetralinyl; C₃₋₁₀ cycloalkyl; 4- to 7-membered        heterocyclyl; or 9- to 11-membered heterobicyclyl, wherein T^(v)        is optionally substituted with one or more R⁹, which are the        same or different;    -   R^(9v) is halogen; —CN; oxo (═O); —C(O)OH; —OH; —S(O)₂NH₂;        —S(O)NH₂; —S(O)₂OH; —S(O)OH; —SH; —NH₂; —NO₂; C₁₋₆ alkyl, or        C₁₋₁₀ heteroalkyl;    -   optionally, one or more of the pairs R^(1v)/R^(1av),        R^(1v)/R^(4v), R^(1v)/R^(6v), R^(1v)/R^(5v), R^(2v)/R^(2v),        R^(2v)/R^(3v), R^(4v)/R^(4av), R^(4v)/R^(5v), R^(5v)/R^(5av),        R^(7v)/R^(7av), R^(7v)/R^(8v), R^(8v)/R^(8av) are joined        together with the atom to which they are attached to form a ring        T;    -   optionally, R^(3v)/R^(3av) are joined together with the nitrogen        atom to which they are attached to form a 4- to 7-membered        heterocycle;    -   and wherein one of R^(1v), R^(1av), R^(2v), R^(2av), R^(3v),        R^(3av), R^(4v), R^(4av), R^(5v), R^(5av), R^(6v), R^(7v),        R^(7av), R^(8v), R^(8av) are substituted with Z⁰ of formula (I).

The carrier Z¹ of formulas (I), (II), (IIaa), (IIab), (IIac), (IIad),(IIb) and (IIba) comprises a covalently bound polymer, preferably apharmaceutically acceptable polymer.

Preferred polymers are selected from 2-methacryloyl-oxyethyl phosphoylcholins, hydrogels, PEG-based hydrogels, poly(acrylic acids),poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyloxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, mannans, pectins, rhamnogalacturonans, starches,hydroxyalkyl starches, hydroxyethyl starches and othercarbohydrate-based polymers, xylans, and copolymers thereof.

Preferably, the carrier Z¹ of formulas (I), (II), (IIaa), (IIab),(IIac), (IIad), (IIb) and (IIba) comprises a poly(oxazoline) or aPEG-based polymer. Most preferably, the carrier Z¹ comprises a PEG-basedpolymer.

In one embodiment the carrier Z¹ of formulas (I), (II), (IIaa), (IIab),(IIac), (IIad), (IIb) and (IIba) may be a hydrogel (as one option for apolymer) which are known in the art. Suitable hydrogels are described inWO-A 2006/003014 or EP-A 1 625 856. If the carrier Z¹ is a hydrogel itis preferred that it is a PEG-based hydrogel as disclosed in WO-A2011/012715 which is incorporated by reference herewith.

Preferably, the carrier Z¹ of formulas (I), (II), (IIaa), (IIab),(IIac), (IIad), (IIb) and (IIba) is a water-soluble carrier.

In one embodiment the carrier Z¹ of formulas (I), (II), (IIaa), (IIab),(IIac), (IIad), (IIb) and (IIba) has the structure of formula (III):

-   -   wherein dashed lines indicate attachment to X⁰ (if the        carrier-linked treprostinil prodrug is of formula (I)), to Q (if        the carrier-linked treprostinil prodrug is of formula (II)), to        Q¹ (if the carrier-linked treprostinil prodrug is of formula        (IIaa) or (IIab)) or to X₁ (if the carrier-linked treprostinil        prodrug is of formula (IIac), (IIad), and (IIb)) or to the rest        of the molecule (if the carrier-linked treprostinil prodrug is        of formula (IIba)), respectively, and    -   wherein each of m, n, and p of formula (III) are independently        an integer ranging of from 5 to 500,    -   and wherein q of formula (III) ranges of from 2 to 32.

In one embodiment the carrier Z¹ of formulas (I), (II), (IIaa), (IIab),(IIac), (IIad), and (IIb) has the structure of formula (IIIa):

-   -   wherein dashed lines indicate attachment to X⁰ (if the        carrier-linked treprostinil prodrug is of formula (I)), to Q (if        the carrier-linked treprostinil prodrug is of formula (II)), to        Q¹ (if the carrier-linked treprostinil prodrug is of formula        (IIaa) or (IIab)) or to X₁ (if the carrier-linked treprostinil        prodrug is of formula (IIac), (IIad), and (IIb)), respectively,        and    -   wherein each of m, n, and p of formula (IIIa) are independently        an integer ranging of from 5 to 500,    -   and wherein q of formula (IIIa) ranges of from 2 to 32.

Preferably, q in formula (III) and (IIIa) is an integer ranging of from2 to 14 and more preferably q of formula (III) and (IIIa) is 6.

Preferably, each of m, n, and p in formula (III) and (IIIa)independently range of from 10 to 250, more preferably from 50 to 150.Preferably, m, n, and p in formula (III) and formula (IIIa) are thesame.

In an alternative embodiment the carrier Z¹ of formulas (I), (II),(IIaa), (IIab), (IIac), (IIad), (IIb), and (IIba) has the structure offormula (IV):

-   -   wherein dashed lines indicate attachment to X⁰ (if the        carrier-linked treprostinil prodrug is of formula (I)), to Q (if        the carrier-linked treprostinil prodrug is of formula (II)), to        Q¹ (if the carrier-linked treprostinil prodrug is of formula        (IIaa) or (IIab)) or to X₁ (if the carrier-linked treprostinil        prodrug is of formula (IIac), (IIad) or (IIb)), or to the rest        of the molecule (if the carrier-linked treprostinil prodrug is        of formula (IIba)), respectively, provided that one of m1, m2 is        1 and wherein the carrier is covalently attached to T in case        m1, m2=0, and    -   wherein each of m, n, and p of formula (IV) are independently an        integer ranging of from 5 to 500 and wherein q of formula (IV)        ranges of from 0 to 14.

Preferably, q in formula (IV) is an integer ranging of from 2 to 6 andmore preferably q of formula (IV) is 2.

Preferably, each of m, n, and p in formula (IV) independently range offrom 10 to 250, more preferably from 50 to 150. Preferably, m, n, and pin formula (IV) are the same.

In another preferred embodiment Z¹ of formulas (I), (II), (IIaa),(IIab), (IIac), (IIad), (IIb), and (IIba) has the structure of formula(V):

Hyp¹ _(mx)-POL^(x)-Hyp²  (V),

-   -   wherein    -   POL^(x) is a polymeric moiety having a molecular weight ranging        from 0.5 kDa to 160 kDa,    -   Hyp¹ and Hyp² are independently a hyperbranched moiety, and    -   mx is 0 or 1.

The polymeric moiety POL^(x) has a molecular weight of from 0.5 kDa to160 kDa, preferably of from 2 kDa to 80 kDa and more preferably of from5 kDa to 40 kDa.

POL^(x) may be selected from the group of polymers consisting of, forexample, polypeptides, 2-methacryloyl-oxyethyl phosphoyl cholins,water-soluble hydrogels, water-soluble PEG-based hydrogels,water-soluble hyaluronic acid-based hydrogels, poly(acrylic acids),poly(acrylates), poly(acrylamides), poly(alkyloxy) polymers,poly(amides), poly(amidoamines), poly(amino acids), poly(anhydrides),poly(aspartamides), poly(butyric acids), poly(glycolic acids),polybutylene terephthalates, poly(caprolactones), poly(carbonates),poly(cyanoacrylates), poly(dimethylacrylamides), poly(esters),poly(ethylenes), poly(ethyleneglycols), poly(ethylene oxides),poly(ethyl phosphates), poly(ethyloxazolines), poly(glycolic acids),poly(hydroxyethyl acrylates), poly(hydroxyethyloxazolines),poly(hydroxymethacrylates), poly(hydroxypropylmethacrylamides),poly(hydroxypropyl methacrylates), poly(hydroxypropyloxazolines),poly(iminocarbonates), poly(lactic acids), poly(lactic-co-glycolicacids), poly(methacrylamides), poly(methacrylates),poly(methyloxazolines), poly(organophosphazenes), poly(ortho esters),poly(oxazolines), poly(propylene glycols), poly(siloxanes),poly(urethanes), poly(vinyl alcohols), poly(vinyl amines),poly(vinylmethylethers), poly(vinylpyrrolidones), silicones, celluloses,carbomethyl celluloses, hydroxypropyl methylcelluloses, chitins,chitosans, dextrans, dextrins, gelatins, hyaluronic acids andderivatives, functionalized hyaluronic acids, mannans, pectins,rhamnogalacturonans, starches, hydroxyalkyl starches, hydroxyethylstarches and other carbohydrate-based polymers, xylans, and copolymersthereof.

The polymeric moiety POL^(x) of formula (V) may comprise a linear orbranched polymer. Preferably, POL^(x) of formula (V) comprises, inparticular consists of a linear polymer.

In one preferred embodiment, POL^(x) of formula (V) comprises, inparticular consists of a PEG-based polymer or a poly(oxazoline)-basedpolymer, more preferably a linear PEG-based polymer. Even morepreferably, POL^(x) of formula (V) consists of a PEG-based linearpolymer.

If m in formula (V) is 0, it is preferred that POL^(x) of formula (V)comprises, preferably consists of a structure of the formulaX1-(OCH₂CH₂)_(p)—O—(CH₂—X2-, wherein n is selected from 2, 3, or 4; p isan integer in the range of from 5 to 2000, preferably p is an integer inthe range of from 10 to 1000, more preferably p is an integer in therange of from 100 to 1000; and X2 is a functional group covalentlylinking POL^(x) and Hyp² of formula (V); and X1 is selected from H, CH₃and C₂H₅.

If m in formula (V) is 1, it is preferred that POL^(x) of formula (V)comprises, preferably consists of a structure of the formulaX3-(CH₂)_(n1)—(OCH₂CH)_(p)—O—(CH₂)_(n2)—X2-, wherein n1 and n2 areindependently selected from 2, 3, and 4; p is an integer in the range offrom 5 to 2000, preferably p is an integer in the range of from 10 to1000, more preferably p is an integer in the range of from 100 to 1000;and X2 and X3 are functional groups covalently linking POL^(x) to Hyp¹and Hyp² of formula (V), respectively.

In a preferred embodiment mx in formula (V) is 0.

In another preferred embodiment, POL^(x) of formula (V) is a polypeptide(or protein), in particular a non-immunogenic polypeptide as describedbelow.

Preferably, the polymeric moiety POL^(x) of formula (V) is a polypeptidewhich comprises at least about 100 amino acid residues, in particularwhich consists of at least about 100 amino acid residues. In a preferredembodiment, amino acids selected from alanine, serine and/or prolineresidues are present, in particular are mainly present, and whichpolypeptide moiety preferably has a random coil conformation atphysiological conditions. It is understood that such a polypeptidemoiety POL^(x) of formula (V) may transiently or temporarily not form arandom coil, for example when present in a lyophilisate or driedcomposition.

A polypeptide moiety POL^(x) of formula (V) may have a random coilconformation with an amino acid sequence consisting of maximally about1000 amino acid residues, preferably of maximally about 900 amino acidresidues, more preferably of maximally about 800 amino acid residues,even more preferably of maximally about 700 amino acid residues,particularly preferably of maximally about 600 amino acid residues.Thus, the amino acid sequence forming random coil conformation mayconsist of maximally about 500 amino acid residues or of maximally about450 amino acid residues.

It is also envisaged herein that the amino acid sequence forming randomcoil conformation may consist of maximally about 1200 and up to about1500 amino acid residues.

Accordingly, the amino acid sequence forming random coil conformationmay consist of about 100 to about 1500 amino acid residues.

In particular embodiments said amino acid sequence forming random coilconformation consists of about 100 to 1000 amino acid residues ascharacterized herein, i.e. comprising alanine, serine and/or proline asmain or unique residues as defined below.

In a preferred embodiment, a polypeptide moiety POL^(x) of formula (V)consists mainly of one, two or three of the amino acid residues alanine,serine and proline, whereby proline residues represent preferably about4% to about 40% of the polypeptide moiety POL^(x) of formula (V). Thealanine and serine residues comprise the remaining at least 60% to 96%of the polypeptide moiety POL^(x) of formula (V). However, as will bedetailed herein below said polypeptide moiety POL^(x) of formula (V) mayalso comprise further amino acids differing from alanine, serine, andproline, i.e. as minor constituents.

The term “minor constituent” as used herein means that maximally 10%(i.e. maximally 10 of 100 amino acids) may be different from alanine,serine and proline, preferably maximally 8% (i.e. maximally 8 of 100amino acids) may be different than alanine, serine and proline, morepreferably maximally 6% (i.e. maximally 6 of 100 amino acids) may bedifferent from alanine, serine and proline, even more preferablymaximally 5% (i.e. maximally 5 of 100 amino acids) may be different fromalanine, serine and proline, particularly preferably maximally 4% (i.e.maximally 4 of 100 amino acids) may be different from alanine, serineand proline, more particularly preferably maximally 3% (i.e. maximally 3of 100 amino acids) may be different from alanine, serine and proline,even more particularly preferably maximally 2% (i.e. maximally 2 of 100amino acids) may be different from alanine, serine and proline and mostpreferably maximally 1% (i.e. maximally 1 of 100 of the amino acids) maybe different from alanine, serine and proline. Said amino acidsdifferent from alanine, serine and proline may be selected from thegroup consisting of different from alanine, serine and proline may beselected from the group of natural or proteinogenic amino-acidscomprising Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met,Phe, Thr, Trp, Tyr, Val, selenocystein, selenomethionin, andhydroxyproline. Minor constituents may also be selected fromnon-naturally occurring amino acids.

The term “at least about 100/150/200/250/300/300/350 (etc) amino acidresidues” is not limited to the concise number of amino acid residuesbut also comprises amino acid stretches that comprise an additional 10%to 20% or comprise 10% to 20% less residues.

For example “at least about 100 amino acid residues” may also encompass80 to 100 and about 100 to 120 amino acid residues without deferringfrom the gist of the present invention.

In one embodiment, the polypeptide moiety POL^(x) of formula (V)comprises a plurality of polymer cassettes wherein said polymercassettes consist of one, two or three of the amino acids selected fromAla, Ser, and Pro and wherein no more than 6 consecutive amino acidresidues are identical and wherein said proline residues constitute morethan 4% and less than 40% of the amino acids of said polypeptide moietyPOL^(x) of formula (V).

A polypeptide moiety POL^(x) of formula (V) may comprise a plurality, inparticular 2, 3, 4, 5 or more of identical polymer cassettes or aplurality of non-identical polymer cassettes. Non-limiting examples ofpolymer cassettes consisting of Ala, Ser and Pro residues are providedherein below; see SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12,SEQ ID NO:13 and SEQ ID NO:14 or peptide fragments or multimers of thesesequences. A polymer cassette may consist of at least 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26,27, 28, 29, 30 or more amino acid residues, wherein each polymercassette comprises (an) Ala, Ser, and Pro residue(s).

In one embodiment, the polymer cassette according to the presentinvention does not comprise more than 100 amino acid residues.Preferably, a polymer cassette as defined herein comprises more thanabout 4%, preferably more than about 5%, even more preferably more thanabout 6%, particularly preferably more than about 8%, more particularlypreferably more than about 10%, even more particularly preferably morethan about 15% and most preferably more than about 20% proline residues.Such polymer cassette as defined herein preferably comprises less thanabout 40% or less than about 35% proline residues.

In one preferred embodiment the polypeptide moiety POL^(x) of formula(V) comprises, in particular consists of formula (a):

Ser_(x)[Ala_(y)Ser_(z)]_(n)  (a),

-   -   which formula further comprises proline residues as defined        herein and wherein    -   x is independently selected from integer 0 to 6,    -   each y is independently selected from integer ranging of from 1        to 6,    -   each z is independently selected from integer ranging of from 1        to 6.    -   n is any integer so that a polypeptide moiety POL^(x) of        formula (V) consists of at least about 100 amino acid residues,        and in particular of at least about 100 to about 3000 amino acid        residues, preferably to about 2000 and more preferably to about        1000 amino acid residues.

In another preferred embodiment, a polypeptide moiety POL^(x) of formula(V) comprises no more than 5 identical consecutive amino acid residues,more preferably no more than 4 identical consecutive amino acid residuesand most preferably no more than 3 identical consecutive amino acidresidues.

As already indicated herein above, a polypeptide moiety POL^(x) offormula (V) comprises in one embodiment proline residues, wherein saidproline residues constitute more than about 4%, preferably more thanabout 5%, even more preferably more than about 6%, particularlypreferably more than about 8%, more particularly preferably more thanabout 10%, even more particularly preferably more than about 15% andmost preferably more than about 20% of the amino acids of POL^(x) offormula (V).

In another preferred embodiment, a polypeptide moiety POL^(x) of formula(V) comprises more than about 4% but less than about 50%, preferablymore than about 10% but less than about 50% and most preferably morethan about 20% but less than about 50% alanine residues of the aminoacids constituting the polypeptide moiety POL^(x) of formula (V).

In a further preferred embodiment, a polypeptide moiety POL^(x) offormula (V) comprises more than about 4% and less than about 50%,preferably more than about 10% but less than about 50% and mostpreferably more than about 20% but less than about 50% serine residuesof the amino acids constituting the polypeptide moiety POL^(x) offormula (V).

Preferably, a polypeptide moiety POL^(x) of formula (V) comprises about35% proline residues, about 50% alanine residues and about 15% serineresidues of the amino acids constituting the polypeptide moiety POL^(x)of formula (V). Alternatively, a polypeptide moiety POL^(x) of formula(V) may comprise about 35% proline residues, about 15% alanine residuesand about 50% serine residues of the amino acids constituting thepolypeptide moiety POL^(x) of formula (V).

Preferably, a polypeptide moiety POL^(x) of formula (V) comprises one ormore of the following alanine-serine polymer cassettes:

SEQ ID NO: 1 AAAASSASSASSSSSAAASA SEQ ID NO: 2 AASAAASSAAASAAAASASSSEQ ID NO: 3 ASASASASASASSAASAASA SEQ ID NO: 4 SAASSSASSSSAASSASAAASEQ ID NO: 5 SSSSAASAASAAAAASSSAS SEQ ID NO: 6 SSASSSAASSSASSSSASAASEQ ID NO: 7 SASASASASASAASSASSAS SEQ ID NO: 8 ASSAAASAAAASSAASASSS

The multimers of these alanine-serine polymer cassettes may form randomcoil conformation in case the resulting amino acid sequence furthercomprises proline residues as defined herein above.

In a preferred embodiment, a polypeptide moiety POL^(x) of formula (V)comprises one or more of the following polymer cassettes:

SEQ ID NO: 9 ASPAAPAPASPAAPAPSAPA SEQ ID NO: 10 AAPASPAPAAPSAPAPAAPSSEQ ID No: 11 APSSPSPSAPSSPSPASPSS SEQ ID NO: 15 SAPSSPSPSAPSSPSPASPS

SEQ ID NO:15 corresponds to the herein provided SEQ ID No:11 in acircularly permuted form, wherein the last serine was removed andanother serine was appended as starting amino acid. As a consequence,multimers of this modified sequence possess essentially the sameinternal repeating unit as multimers of the non-modified sequence,except for the very first and the very last residue. Accordingly, SEQ IDNO:15 may be considered as an example of a further polymer cassette fora polypeptide moiety POL^(x) of formula (V). It is clear for the personskilled in the art that also other polymer cassettes and (shorter)peptide fragments or circularly permuted versions of the herein providedamino acid polymers may be used as polymer cassettes for a polypeptidemoiety POL^(x) of formula (V).

Yet, even further and illustrative amino acid polymers forming randomcoil conformation may comprise amino acid sequences that may be selectedfrom the group consisting of the following sequences:

SEQ ID NO: 12 SSPSAPSPSSPASPSPSSPA SEQ ID NO: 13AASPAAPSAPPAAASPAAPSAPPA SEQ ID NO: 14 ASAAAPAAASAAASAPSAAA

Therefore, preferred polymer cassettes for a polypeptide moiety POL^(x)of formula (V) are selected from the following sequences:

(SEQ ID NO: 9) ASPAAPAPASPAAPAPSAPA, (SEQ ID NO: 10)AAPASPAPAAPSAPAPAAPS, (SEQ ID NO: 11) APSSPSPSAPSSPSPASPSS,(SEQ ID NO: 12) SSPSAPSPSSPASPSPSSPA, (SEQ ID NO: 13)AASPAAPSAPPAAASPAAPSAPPA, and (SEQ ID NO: 14) ASAAAPAAASAAASAPSAAA;

-   -   or circular permuted versions or (a) multimer(s) of these        sequences as a whole or parts of these sequences.

Again, also (a) peptide fragment(s) or (a) multimer(s) or circularlypermuted versions of these sequences and the sequences provided hereinabove may be employed in context of the present invention as polymercassettes for a polypeptide moiety POL^(x) of formula (V). The personskilled in the art is readily in a position to generate further aminoacid polymer cassettes that form random coil conformation underphysiological conditions and are constituted of mainly alanine, serine,and proline as defined herein. Such other and further examples of randomcoil conformation forming amino acid polymer cassettes to be used for apolypeptide moiety POL^(x) of formula (V) may, inter alia, comprisecombinations and/or peptide fragments or circularly permuted versions ofthe specific polymer cassettes shown above.

Accordingly, the exemplified polymer cassettes may also provide forindividual peptide fragments which may be newly combined to form furtherpolymer cassettes.

In accordance with the above, a polypeptide moiety POL^(x) of formula(V) may comprise a multimer of sequences consisting of either one of theamino acid sequences with SEQ ID NO:9, 10, 11, 12, 13 or 14 as disclosedherein above or may comprise a multimer of sequences consisting of morethan one of amino acid sequences SEQ ID NOs:9, 10, 11, 12, 13 and 14.Furthermore, it is envisaged that also peptide fragments or circularlypermuted versions of these exemplified sequences may be used to build upfurther polymer cassettes of a polypeptide moiety POL^(x) of formula(V).

In another embodiment, a polypeptide moiety POL^(x) of formula (V) maycomprise a multimer of sequences consisting of a (circular) permutationof the amino acid sequence selected from the group consisting of SEQ IDNO:9, 10, 11, 12, 13, 14, 15 or (a) multimers(s) of these (circular)permutated sequences.

In yet another embodiment, a polypeptide moiety POL^(x) of formula (V)may comprise a multimer consisting of a peptide fragment/part of theamino acid sequence selected from the group consisting of SEQ ID NOs: 9,10, 12, 13, 14, 15 or (a) multimers(s) of these exemplified polymercassettes.

Peptide fragments of these sequences to be employed for the generationof a polypeptide moiety POL^(x) of formula (V) may consist of at least3, preferably of at least 4, more preferably of at least 5, even morepreferably of at least 6, still more preferably of at least 8,particularly preferably of at least 10, more particularly preferably ofat least 12, even more particularly preferably of at least 14,preferably of at least 6, still more preferably of at least 8,particularly preferably of at least 10, more particularly preferably ofat least 12, even more particularly preferably of at least 14, even moreparticularly preferably of at least 16, and most preferably of at least18 consecutive amino acids of the amino acid sequence selected from thegroup consisting of said SEQ ID NOs: 9, 10, 11, 12, 13 and 14.

For example, individual peptide fragments of the inventive polymercassettes may be combined to further individual polymer cassettes aslong as the above-identified rules for the overall distribution andamount of alanine, serine and proline are respected. Again, thesepolymer cassettes may also comprise further amino acid residues, howeveronly as minimal or minor constituents, i. e. maximally 10%, preferablymaximally 2% of the individual polymer cassette. POL^(x) of formula (V)moieties comprising polymer cassettes consist, in one embodiment of thepresent invention, of at least about 100 amino acid residues. Individualpolymer cassettes may be combined in order to form longer random coilforming amino acid polymers, whereby a maximal length of a polypeptidemoiety POL^(x) of formula (V) is about 3000 amino acids.

Preferably, POL^(x) of formula (V) is covalently linked to Hyp¹ and Hyp²of formula (V), in particular by a permanent linkage, more preferably bya permanent amide linkage.

In the carrier-linked treprostinil prodrugs of the present inventionfunctional groups of Hyp¹ and Hyp² of formula (V) are connected to amoiety X⁰ of formula (I), to a moiety Q (if the carrier-linkedtreprostinil prodrug is of formula (II)), to a moiety Q¹ (if thecarrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)) orto a moiety X₁ (if the carrier-linked treprostinil prodrug is of formula(IIac), (IIad), or (IIb)), or to the rest of the molecule (if thecarrier-linked treprostinil prodrug is of formula (IIba)), respectively.

The hyperbranched moieties Hyp¹ and Hyp² of formula (V) are eachindependently selected from the group comprising, in particularconsisting of, in bound form glycerol, pentaerythritol,dipentaerythritol, tripentaerythritol, hexaglycerine, sucrose, sorbitol,fructose, mannitol, glucose, cellulose, amyloses, starches, hydroxyalkylstarches, polyvinylalcohols, dextranes, hyualuronans, dilysine,trilysine, tetralysine, pentalysine, hexalysine, heptalysine,octalysine, nonalysine, decalysine, undecalysine, dodecalysine,tridecalysine, tetradecalysine, pentadecalysine, hexadecalysine,heptadecalysine, octadecalysine, nonadecalysine, triornithine,tetraornithine, pentaornithine, hexaornithine, heptaornithine,octaornithine, nonaornithine, decaornithine, undecaornithine,dodecaornithine, tridecaornithine, tetradecaornithine,pentadecaornithine, hexadecaornithine, heptadecaornithine,octadecaornithine, nonadecaornithine, tridiaminobutyric acid,tetradiaminobutyric acid, pentadiaminobutyric acid, hexadiaminobutyricacid, heptadiaminobutyric acid, octadiaminobutyric acid,nonadiaminobutyric acid, decadiaminobutyric acid, undecadiaminobutyricacid, dodecadiaminobutyric acid, tridecadiaminobutyric acid,tetradecadiaminobutyric acid, pentadecadiaminobutyric acid,hexadecadiaminobutyric acid, heptadecadiaminobutyric acid,octadecadiaminobutyric acid, nonadecadiaminobutyric acid, di(glutamicacid), tri(glutamic acid), tetra(glutamic acid), penta(glutamic acid),hexa(glutamic acid), hepta(glutamic acid), octa(glutamic acid),nona(glutamic acid), deca(glutamic acid), undeca(glutamic acid),dodeca(glutamic acid), trideca(glutamic acid), tetradeca(glutamic acid),pentadeca(glutamic acid), hexadeca(glutamic acid), heptadeca(glutamicacid), octadeca(glutamic acid), nonadeca(glutamic acid), di(asparticacid), tri(aspartic acid), tetra(aspartic acid), penta(aspartic acid),hexa(aspartic acid), hepta(aspartic acid), octa(aspartic acid),nona(aspartic acid), deca(aspartic acid), undeca(aspartic acid),dodeca(aspartic acid), trideca(aspartic acid), tetradeca(aspartic acid),pentadeca(aspartic acid), hexadeca(aspartic acid), heptadeca(asparticacid), octadeca(aspartic acid), nonadeca(aspartic acid),polyethyleneimines, and low-molecular weight PEI.

In a preferred embodiment, the hyperbranched moieties Hyp¹ and Hyp² offormula (V) are each independently selected from the group comprising,in particular consisting of, in bound form dilysine, trilysine,tetralysine, pentalysine, hexalysine, heptalysine, octalysine,nonalysine, decalysine, undecalysine, dodecalysine, tridecalysine,tetradecalysine, pentadecalysine, hexadecalysine, heptadecalysine,octadecalysine, nonadecalysine, triornithine, tetraornithine,pentaornithine, hexaornithine, heptaornithine, octaornithine,nonaornithine, decaornithine, undecaornithine, dodecaornithine,tridecaornithine, tetradecaornithine, pentadecaornithine,hexadecaornithine, heptadecaornithine, octadecaornithine,nonadecaornithine, tridiaminobutyric acid, tetradiaminobutyric acid,pentadiaminobutyric acid, hexadiaminobutyric acid, heptadiaminobutyricacid, octadiaminobutyric acid, nonadiaminobutyric acid,decadiaminobutyric acid, undecadiaminobutyric acid, dodecadiaminobutyricacid, tridecadiaminobutyric acid, tetradecadiaminobutyric acid,pentadecadiaminobutyric acid, hexadecadiaminobutyric acid,heptadecadiaminobutyric acid, octadecadiaminobutyric acid,nonadecadiaminobutyric acid, di(glutamic acid), tri(glutamic acid),tetra(glutamic acid), penta(glutamic acid), hexa(glutamic acid),hepta(glutamic acid), octa(glutamic acid), nona(glutamic acid),deca(glutamic acid), undeca(glutamic acid), dodeca(glutamic acid),trideca(glutamic acid), tetradeca(glutamic acid), pentadeca(glutamicacid), hexadeca(glutamic acid), heptadeca(glutamic acid),octadeca(glutamic acid), nonadeca(glutamic acid), di(aspartic acid),tri(aspartic acid), tetra(aspartic acid), penta(aspartic acid),hexa(aspartic acid), hepta(aspartic acid), octa(aspartic acid),nona(aspartic acid), deca(aspartic acid), undeca(aspartic acid),dodeca(aspartic acid), trideca(aspartic acid), tetradeca(aspartic acid),pentadeca(aspartic acid), hexadeca(aspartic acid), heptadeca(asparticacid), octadeca(aspartic acid), nonadeca(aspartic acid),polyethyleneimines, and low-molecular weight PEI.

More preferably, the hyperbranched moieties Hyp¹ and Hyp² of formula (V)are independently selected from the group comprising, more preferablyconsisting of, in bound form, trilysine, tetralysine, pentalysine,hexalysine, heptalysine, octalysine, nonalysine, decalysine,undecalysine, dodecalysine, tridecalysine, tetradecalysine,pentadecalysine, hexadecalysine, and heptadecalysine, even morepreferably Hyp¹ and Hyp² are independently comprising, preferablyconsisting of, in bound form, trilysine, heptalysine or pentadecalysine.

More preferably, Hyp¹ and Hyp² of formula (V) are independently selectedfrom any one of the following structures:

-   -   wherein    -   dashed lines marked with an asterisk indicate attachment to        POL^(x) of formula (V),    -   unmarked dashed lines indicate attachment to X⁰ (if the        carrier-linked treprostinil prodrug is of formula (I)), to Q (if        the carrier-linked treprostinil prodrug is of formula (II)), to        Q¹ (if the carrier-linked treprostinil prodrug is of formula        (IIaa) or (IIab)) or to X₁ (if the carrier-linked treprostinil        prodrug is of formula (IIac), (IIad), or (IIb)), or to the rest        of the molecule (if the carrier-linked treprostinil prodrug is        of formula (IIba)), respectively, and    -   qx is an integer of from 0 to 15, preferably 3 to 7, and even        more preferably 6.

Preferably, Hyp¹ and Hyp² of formula (V) are each a heptalysinyl group,in particular Hyp¹ and Hyp² of formula (V) each have the structure offormula (ii-x) above.

Preferably, Hyp¹ and Hyp² of formula (V) have the same structure.

Functional groups of Hyp¹ and Hyp² of formula (V) serve as attachmentpoints for direct linkage of Hyp¹ and Hyp² of formula (V) to X⁰ (if thecarrier-linked treprostinil prodrug is of formula (I)), to Q (if thecarrier-linked treprostinil prodrug is of formula (II)), to Q¹ (if thecarrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)) orto X₁ (if the carrier-linked treprostinil prodrug is of formula (IIac),(IIad), or (IIb)), or to the rest of the molecule (if the carrier-linkedtreprostinil prodrug is of formula (IIba)), respectively. Remainingfunctional groups which are not connected to X⁰, Q, Q¹ or X₁,respectively, may, independently of each other, be capped with suitablecapping reagents or may optionally be connected to at least onetargeting moiety, in particular through permanent linkages

Therefore, in the water-soluble carrier-linked prodrugs of the presentinvention the hyperlinked moieties Hyp¹ and Hyp² of formula (V) areconnected to POL^(x) of formula (V) and functional groups of Hyp¹ andHyp² of formula (V) are connected to X⁰ of formula (I), to Q (if thecarrier-linked treprostinil prodrug is of formula (II)), to Q¹ (if thecarrier-linked treprostinil prodrug is of formula (IIaa) or (IIab)) orto X₁ (if the carrier-linked treprostinil prodrug is of formula (IIac),(IIad), or (IIb)), or to the rest of the molecule (if the carrier-linkedtreprostinil prodrug is of formula (IIba)), respectively, permanentlinkages, targeting moieties and/or capping groups.

In a preferred embodiment, all functional groups of the hyperbranchedmoieties Hyp¹ and Hyp² of formula (V) are connected to X⁰ of formula(I), to Q (if the carrier-linked treprostinil prodrug is of formula(II)), to Q¹ (if the carrier-linked treprostinil prodrug is of formula(IIaa) or (IIab)) or to X₁ (if the carrier-linked treprostinil prodrugis of formula (IIac), (IIad) or (IIb)), or to the rest of the molecule(if the carrier-linked prodrug is of formula (IIba)), respectively.

Preferably, the hyperbranched moieties Hyp¹ and Hyp² of formula (V) haveindependently a molecular weight in the range of from 0.1 kDa to 4 kDa,more preferably 0.4 kDa to 2 kDa. Preferably, the hyperbranched moietiesHyp¹ and Hyp² of formula (V) have each independently at least 3branchings and are each independently conjugated to at least 4 X⁰, Q,Q¹, X₁ or the rest of the molecule, respectively, permanent linkages,and/or capping groups and each independently have at most 63 branchingsand are each independently at most conjugated to 64 X⁰, Q, Q¹, X¹ or therest of the molecule, respectively, permanent linkages, and/or cappinggroups. It is preferred that the hyperbranched moieties Hyp¹ and Hyp² offormula (V) have each independently at least 7 branchings and are eachindependently conjugated to at least 8 X⁰, Q, Q¹, X₁ or the rest of themolecule, respectively, permanent linkages, and/or capping groups andhave each independently at most 31 branchings and are each independentlyat most conjugated to 32 X⁰, Q, Q¹, X¹ or the rest of the molecule,respectively, permanent linkages, and/or capping groups.

Preferably, the hyperbranched moieties Hyp¹ and Hyp² of formula (V) areeach independently a hyperbranched polypeptide. Preferably, suchhyperbranched polypeptide comprises lysine in bound form. Preferably,each hyperbranched moiety Hyp¹ and Hyp² of formula (V) independentlyhave a molecular weight in the range of from 0.1 kDa to 4 kDa, inparticular 0.4 kDa to 2 kDa.

Preferably, mx is 0 and POL-Hyp²- of formula (V) is selected from thefollowing structures:

-   -   wherein    -   dashed lines indicate attachment to X⁰ (if the carrier-linked        treprostinil prodrug is of formula (I)), to Q (if the        carrier-linked treprostinil prodrug is of formula (II)), to Q¹        (if the carrier-linked treprostinil prodrug is of formula (IIaa)        or (IIab)) or to X¹ (if the carrier-linked treprostinil prodrug        is of formula (IIac) or (IIad)), respectively, provided that one        of m1, m2 of formula (I) is 1 and wherein the carrier is        covalently attached to T of formula (I) in case m1, m2=0,    -   px is an integer of from 5 to 2000, preferably 10 to 1000, in        particular 100 to 1000, and    -   qx is an integer of from 0 to 15, preferably 3 to 7, more        preferably, qx is 6.

In another preferred embodiment Z¹ of formula (I), (II), (IIaa), (IIab),(IIac), (IIad), (IIb) and (IIba) has the structure of formula (VI):

BA-Hyp^(y))_(n)  (VI),

-   -   wherein    -   B is a branching core,    -   each A is independently a poly(ethylene glycol)-based polymeric        chain,    -   each Hyp^(y) is independently a branched moiety, and    -   n is an integer of from 3 to 32;

In a preferred embodiment, the branching core B of formula (VI)comprises, preferably consists of a moiety selected from:

-   -   a polyalcohol comprising at least 2 hydroxyl groups (preferably        further comprising a functional group, which is preferably an        additional amino group or a carboxylic acid group, more        preferably an additional carboxylic acid group),    -   preferably B is selected from glycerol, pentaerythritol,        dipentaerythritol, tripentaerythritol, hexaglycerine, sucrose,        sorbitol, fructose, mannitol, glucose, cellulose, amyloses,        starches, hydroxyalkyl starches, polyvinylalcohols, dextranes,        and hyualuronans, erythritol, threitol, arabitol, xylitol,        ribitol, dulcitol, iditol; more preferably from glycerol,        pentaerythritol, dipentaerythritol, tripentaerythritol,        hexaglycerine, sucrose, sorbitol, fructose, mannitol, glucose,        cellulose, amyloses, starches, hydroxyalkyl starches,        polyvinylalcohols, dextranes, and hyualuronans.    -   or a polyamine comprising at least 2 amine groups (preferably        further comprising a functional group, which is preferably an        additional hydroxyl group or a carboxylic acid group, more        preferably a carboxylic acid group),    -   preferably selected from ornithine, diornithine, triornithine,        tetraornithine, pentaornithine, hexaornithine, heptaornithine,        octaornithine, nonaornithine, decaornithine, undecaornithine,        dodecaornithine, tridecaornithine, tetradecaornithine,        pentadecaornithine, hexadecaornithine, heptadecaornithine,        octadecaornithine, nonadecaornithine, diaminobutyric acid,        di(diaminobutyric acid), tri(diaminobutyric acid),        tetra(diaminobutyric acid), penta(diaminobutyric acid),        hexa(diaminobutyric acid), hepta(diaminobutyric acid),        octa(diaminobutyric acid), nona(diaminobutyric acid),        deca(diaminobutyric acid), undeca(diaminobutyric acid),        dodeca(diaminobutyric acid), trideca(diaminobutyric acid),        tetradeca(diaminobutyric acid), pentadeca(diaminobutyric acid),        hexadeca(diaminobutyric acid), heptadeca(diaminobutyric acid),        octadeca(diaminobutyric acid), nonadeca(diaminobutyric acid),        lysine, dilysine, trilysine, tetralysine, pentalysine,        hexalysine, heptalysine, octalysine, nonalysine, decalysine,        undecalysine, dodecalysine, tridecalysine, tetradecalysine,        pentadecalysine, hexadecalysine, heptadecalysine,        octadecalysine, nonadecalysine, oligolysines,        polyethyleneimines, and polyvinylamines;    -   wherein the polyalcohol or polyamine is in bound form.

In a preferred embodiment, the branching core B of formula (VI)comprises, preferably consists of pentaerithritol.

Preferably, a poly(ethylene glycol)-based polymeric chain A connected tothe branching core B of formula (VI) consists of a linear PEG chain, ofwhich one terminus is connected to B of formula (VI) and the otherterminus is connected to Hyp^(y) of formula (VI). It is understood thata PEG-based chain A of formula (VI) may optionally be terminated in caseof a branched PEG chain and/or may optionally be interrupted in case ofa branched or linear PEG chain by alkyl or aryl groups and mayoptionally be substituted with heteroatoms and/or functional groups.

Each sub-structure A-Hyp^(y) of formula (VI) extending from thebranching core B of formula (VI) may be independently of each other thesame or different sub-structures A-Hyp^(y). In a preferred embodiment,the all sub-structures A-Hyp^(y) of formula (VI) are the same.

Each A and each Hyp^(y) of formula (VI) may be independently selectedfrom the other moieties A and Hyp^(y). Preferably, all sub-structuresA-Hyp^(y) connected to B of formula (VI) have an identical structure.

Preferably, the PEG-based polymeric chains A of formula (VI) areconnected to B through permanent linkages.

n of formula (VI) is an integer from 3 to 32. Preferably, n is aninteger from 3 to 16, more preferably n is an integer from 4 to 8 andmost preferably n is 4.

In a preferred embodiment n of formula (VI) is 4 and m is 2.

In one embodiment, a PEG-based polymeric chain A of formula (VI) isselected from a linear or branched PEG-based polymeric chain.Preferably, A is a linear PEG-based polymeric chain.

Preferably, each A of formula (VI) is independently selected from theformula

—X3-(CH₂)_(n1)—(OCH₂CH₂)_(p)—O—(CH₂)_(n2)—X2-,

-   -   wherein    -   n1 and n2 are independently selected from 1, 2, 3, and 4,        preferably from 1, 2, and 3;    -   p is an integer in the range of from 5 to 2000, preferably p is        an integer in the range of from 10 to 1000, more preferably p is        an integer in the range of from 100 to 1000; and    -   X3 and X2 are independently functional groups covalently linked        to B or Hyp^(y), respectively.

Preferably, a linkage between a moiety A and a moiety Hyp^(y) of formula(VI) is a permanent linkage, more preferably a permanent linkagecomprising a linkage group comprising, in particular consisting of agroup selected from amine groups, amide groups, carbamate groups,thioether groups, ether groups, and most preferably a permanent linkagebetween a moiety A and a moiety Hyp^(y) of formula (VI) is an amidelinkage.

In a preferred embodiment, a sub-structure B (A)_(n) formula (VI) is amulti-arm PEG derivative as, for instance, detailed in the products listof JenKem Technology, USA (accessed by download fromhttp://jenkemusa.net/pegproducts2.aspx on Mar. 8, 2011), such as a4-arm-PEG derivative, in particular comprising a pentaerythritol core,an 8-arm-PEG derivative comprising a hexaglycerin core, and an 8-arm-PEGderivative comprising a tripentaerythritol core. Most preferred aresub-structures B (A)_(n) of formula (VI) comprising, in particularconsisting of, moieties selected from:

a 4-arm PEG Amine comprising a pentaerythritol core:

with n ranging from 400 to 2000;

a 4-arm PEG Carboxyl comprising a pentaerythritol core:

with n ranging from 400 to 2000;

an 8-arm PEG Amine comprising a hexaglycerin core:

with n ranging from 400 to 2000 and

R=hexaglycerin core structure;

an 8-arm PEG Carboxyl comprising a hexaglycerin core:

with n ranging from 400 to 2000 and

R=hexaglycerin core structure;

an 8-arm PEG Amine comprising a tripentaerythritol core:

with n ranging from 400 to 2000

and R=tripentaerythritol core structure;

and an 8-arm PEG Carboxyl comprising a tripentaerythritol core:

with n ranging from 400 to 2000 and

R=tripentaerythritol core structure;

a 6-arm PEG Amine comprising a sorbitol or dipentaerythritol core:

with n ranging from 400 to 2000 and

R=sorbitol or dipentaerythritol;

an 8-arm PEG Carboxyl comprising a sorbitol or dipentaerythritol core:

with n ranging from 400 to 2000 and

R=sorbitol or dipentaerythritol;

an 8-arm PEG Amine comprising a sorbitol or dipentaerythritol core:

with n ranging from 400 to 2000

and R=sorbitol or dipentaerythritol;

and an 8-arm PEG Carboxyl comprising a sorbitol or dipentaerythritolcore:

with n ranging from 400 to 2000 and

R=sorbitol or dipentaerythritol;

each in bound form.

Also preferred are sub-structures B (

_(n) of the following formulas:

a 4-arm PEG Amine comprising a pentaerythritol core:

with n ranging from 400 to 2000;

a 4-arm PEG Carboxyl comprising a pentaerythritol core:

with n ranging from 400 to 2000;

an 8-arm PEG Amine comprising a hexaglycerin core:

with n ranging from 400 to 2000 and

R=hexaglycerin core structure;

an 8-arm PEG Carboxyl comprising a hexaglycerin core:

with n ranging from 400 to 2000 and

R=hexaglycerin core structure;

an 8-arm PEG Amine comprising a tripentaerythritol core:

with n ranging from 400 to 2000

and R=tripentaerythritol core structure;

and an 8-arm PEG Carboxyl comprising a tripentaerythritol core:

with n ranging from 400 to 2000 and

R=tripentaerythritol core structure;

a 6-arm PEG Amine comprising a sorbitol or dipentaerythritol core:

with n ranging from 400 to 2000 and

R=sorbitol or dipentaerythritol;

a 6-arm PEG Carboxyl comprising a sorbitol or dipentaerythritol core:

with n ranging from 400 to 2000 and

R=sorbitol or dipentaerythritol;

a 6-arm PEG Amine comprising a sorbitol or dipentaerythritol core:

with n ranging from 400 to 2000

and R=sorbitol or dipentaerythritol;

and a 6-arm PEG Carboxyl comprising a sorbitol or dipentaerythritolcore:

with n ranging from 400 to 2000 and

R=sorbitol or dipentaerythritol;

each in bound form.

Also preferred are sub-structures B

_(n) of formula (VI) comprising, in particular consisting of, moietiesselected from:

a 4-arm PEG Amine comprising a pentaerythritol core:

with n ranging from 20 to 500;

a 4-arm PEG Carboxyl comprising a pentaerythritol core:

with n ranging from 20 to 500;

an 8-arm PEG Amine comprising a hexaglycerin core:

with n ranging from 20 to 500; and

R=hexaglycerin core structure;

an 8-arm PEG Carboxyl comprising a hexaglycerin core:

with n ranging from 20 to 500; and

R=hexaglycerin core structure;

an 8-arm PEG Amine comprising a tripentaerythritol core:

with n ranging from 20 to 500;

and R=tripentaerythritol core structure;

and an 8-arm PEG Carboxyl comprising a tripentaerythritol core:

with n ranging from 20 to 500; and

R=tripentaerythritol core structure;

a 6-arm PEG Amine comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500; and

R=sorbitol or dipentaerythritol;

an 8-arm PEG Carboxyl comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500; and

R=sorbitol or dipentaerythritol;

an 8-arm PEG Amine comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500;

and R=sorbitol or dipentaerythritol;

and an 8-arm PEG Carboxyl comprising a sorbitol or dipentaerythritolcore:

with n ranging from 20 to 500; and

R=sorbitol or dipentaerythritol;

each in bound form.

Also preferred are sub-structures B (

_(n) of the following formulas:

a 4-arm PEG Amine comprising a pentaerythritol core:

with n ranging from 20 to 500;

a 4-arm PEG Carboxyl comprising a pentaerythritol core:

with n ranging from 20 to 500;

an 8-arm PEG Amine comprising a hexaglycerin core:

with n ranging from 20 to 500; and

R=hexaglycerin core structure;

an 8-arm PEG Carboxyl comprising a hexaglycerin core:

with n ranging from 20 to 500; and

R=hexaglycerin core structure;

an 8-arm PEG Amine comprising a tripentaerythritol core:

with n ranging from 20 to 500;

and R=tripentaerythritol core structure;

and an 8-arm PEG Carboxyl comprising a tripentaerythritol core:

with n ranging from 20 to 500; and

R=tripentaerythritol core structure;

a 6-arm PEG Amine comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500; and

R=sorbitol or dipentaerythritol;

a 6-arm PEG Carboxyl comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500; and

R=sorbitol or dipentaerythritol;

a 6-arm PEG Amine comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500;

and R=sorbitol or dipentaerythritol;

and a 6-arm PEG Carboxyl comprising a sorbitol or dipentaerythritolcore:

with n ranging from 20 to 500; and

R=sorbitol or dipentaerythritol;

each in bound form.

In a preferred embodiment, the molecular weight of a sub-structureB-(A)_(n) of formula (VI) ranges from 1 kDa to 80 kDa, more preferably 1kDa to 40 kDa and even more preferably 10 kDa to 40 kDa. It isunderstood that the terminal amine groups or carboxyl groups,respectively, are used for conjugation to a moiety Hyp^(y) of formula(VI).

Functional groups of a moiety Hyp^(y) of formula (VI) are connected tomoieties X⁰ of formula (I), to Q (if the carrier-linked treprostinilprodrug is of formula (II)), to Q¹ (if the carrier-linked treprostinilprodrug is of formula (IIaa) or (IIab)) or to X₁ (if the carrier-linkedtreprostinil prodrug is of formula (IIac), (IIad), or (IIb)), or to therest of the molecule (if the carrier-linked treprostinil prodrug is offormula (IIba)), respectively.

In a preferred embodiment, a moiety Hyp^(y) of formula (VI) is connectedto a moiety X⁰ of formula (I), to Q (if the carrier-linked treprostinilprodrug is of formula (II)), to Q¹ (if the carrier-linked treprostinilprodrug is of formula (IIaa) or (IIab)) or to X₁ (if the carrier-linkedtreprostinil prodrug is of formula (IIac), (IIad), or (IIb)), or to therest of the molecule (if the carrier-linked treprostinil prodrug is offormula (IIba)), respectively, through a functional group selected fromamide groups, carbamate groups, ester groups, ether groups, aminegroups, thioether groups. Preferably, a moiety Hyp^(y) of formula (VI)is connected to a moiety X⁰ of formula (I), to a moiety Q of formula(II), to a moiety Q¹ of either formula (IIaa) or (IIab) or to a moietyX₁ of formula (IIac), (IIad), or (IIb), or to the rest of the moleculeof formula (IIba), respectively, through amide groups, thioether groupsand/or ether groups, even more preferably through amide groups.

Optionally, functional groups of a moiety Hyp^(y) of formula (VI) whichare not connected to a moiety X⁰ of formula (I), to a moiety Q offormula (II), to a moiety Q¹ of either formula (IIaa) or (IIab) or to amoiety X₁ of formula (IIac), (IIad). (IIb), or to the rest of themolecule of formula (IIba) respectively, may be capped with suitablecapping reagents and/or may optionally be connected to at least onetargeting moiety, in particular through permanent linkages. Therefore, amoiety Hyp^(y) of formula (VI) may be connected to a moiety X⁰ offormula (I), to a moiety Q of formula (II), to a moiety Q¹ of eitherformula (IIaa) or (IIab) or to a moiety X₁ of formula (IIac), (IIad),(IIb), or to the rest of the molecule of formula (IIba), respectively,capping moieties and/or targeting moieties. Preferably, functionalgroups of a moiety Hyp^(y) of formula (VI) are connected to a moiety X⁰of formula (I), to a moiety Q of formula (II), to a moiety Q¹ of eitherformula (IIaa) or (IIab) or to a moiety X₁ of formula (IIac), (IIad), or(IIb), or to the rest of the molecule of formula (IIba), respectively,and are not connected to capping moieties and/or targeting moieties.Targeting moieties, if present, may be conjugated to a moiety Hyp^(y) offormula (VI) either directly or indirectly through spacer moieties.

Examples of suitable capping moieties are linear, branched or cyclicC₁₋₈ alkyl groups.

In one embodiment, each branched moiety Hyp^(y) of formula (VI) isdirectly or indirectly connected to at least two moieties X⁰ of formula(I), to at least two moieties Q of formula (II), to at least twomoieties Q¹ of either formula (IIaa) or (IIab) or to at least twomoieties X¹ of formula (IIac), (IIad), or (IIb), or to the rest of themolecule of formula (IIba), respectively. More preferably, each branchedmoiety Hyp^(y) of formula (VI) is directly or indirectly connected to atleast three moieties X⁰ of formula (I), to at least three moieties Q offormula (II), to at least three moieties Q¹ of either formula (IIaa) or(IIab) or to at least three moieties X₁ of formula (IIac), (IIad), or(IIb), or to the rest of the molecule of formula (IIba), respectively.Most preferably, each branched moiety Hyp^(y) of formula (VI) isdirectly or indirectly connected to at least four moieties X⁰ of formula(I), to at least four moieties Q of formula (II), to at least fourmoieties Q¹ of either formula (IIaa) or (IIab) or to at least fourmoieties X₁ of formula (IIac), (IIad) or (IIb), or to the rest of themolecule of formula (IIba), respectively.

The branched moiety Hyp^(y) of formula (VI) comprises, preferablyconsists of a moiety in bound form selected from:

-   -   a polyalcohol in bound form comprising at least 2 hydroxyl        groups (preferably further comprising a functional group, which        is preferably an additional hydroxyl group or a carboxylic acid        group, more preferably an additional hydroxyl group),    -   preferably selected from glycerol, pentaerythritol,        dipentaerythritol, tripentaerythritol, hexaglycerine, sucrose,        sorbitol, fructose, mannitol, glucose, cellulose, amyloses,        starches, hydroxyalkyl starches, polyvinylalcohols, dextranes,        and hyualuronans, erythritol, threitol, arabitol, xylitol,        ribitol, dulcitol, iditol; more preferably from glycerol,        pentaerythritol, dipentaerythritol, tripentaerythritol,        hexaglycerine, sucrose, sorbitol, fructose, mannitol, glucose,        cellulose, amyloses, starches, hydroxyalkyl starches,        polyvinylalcohols, dextranes, and hyualuronans;    -   or a polyamine in bound form comprising at least 2 amine groups        (preferably further comprising a functional group, which is        preferably an additional amine group or a carboxylic acid group,        more preferably a carboxylic acid group),    -   preferably selected from ornithine, diornithine, triornithine,        tetraornithine, pentaornithine, hexaornithine, heptaornithine,        octaornithine, nonaornithine, decaornithine, undecaornithine,        dodecaornithine, tridecaornithine, tetradecaornithine,        pentadecaornithine, hexadecaornithine, heptadecaornithine,        octadecaornithine, nonadecaornithine, diaminobutyric acid,        di(diaminobutyric acid), tri(diaminobutyric acid),        tetra(diaminobutyric acid), penta(diaminobutyric acid),        hexa(diaminobutyric acid), hepta(diaminobutyric acid),        octa(diaminobutyric acid), nona(diaminobutyric acid),        deca(diaminobutyric acid), undeca(diaminobutyric acid),        dodeca(diaminobutyric acid), trideca(diaminobutyric acid),        tetradeca(diaminobutyric acid), pentadeca(diaminobutyric acid),        hexadeca(diaminobutyric acid), heptadeca(diaminobutyric acid),        octadeca(diaminobutyric acid), nonadeca(diaminobutyric acid),        lysine, dilysine, trilysine, tetralysine, pentalysine,        hexalysine, heptalysine, octalysine, nonalysine, decalysine,        undecalysine, dodecalysine, tridecalysine, tetradecalysine,        pentadecalysine, hexadecalysine, heptadecalysine,        octadecalysine, nonadecalysine, oligolysines, triornithine,        tetraornithine, pentaornithine, hexaornithine, heptaornithine,        octaornithine, nonaornithine, decaornithine, undecaornithine,        dodecaornithine, tridecaornithine, tetradecaornithine,        pentadecaornithine, hexadecaornithine, heptadecaornithine,        octadecaornithine, nonadecaornithine, tridiaminobutyric acid,        tetradiaminobutyric acid, pentadiaminobutyric acid,        hexadiaminobutyric acid, heptadiaminobutyric acid,        octadiaminobutyric acid, nonadiaminobutyric acid,        decadiaminobutyric acid, undecadiaminobutyric acid,        dodecadiaminobutyric acid, tridecadiaminobutyric acid,        tetradecadiaminobutyric acid, pentadecadiaminobutyric acid,        hexadecadiaminobutyric acid, heptadecadiaminobutyric acid,        octadecadiaminobutyric acid, nonadecadiaminobutyric acid,    -   or a polycarboxylate in bound form comprising at least 2        carboxylate groups (preferably further comprising a functional        group, which is preferably an additional amino group or a        carboxylic acid group, more preferably an additional carboxylic        acid group),    -   preferably selected from di(glutamic acid), tri(glutamic acid),        tetra(glutamic acid), penta(glutamic acid), hexa(glutamic acid),        hepta(glutamic acid), octa(glutamic acid), nona(glutamic acid),        deca(glutamic acid), undeca(glutamic acid), dodeca(glutamic        acid), trideca(glutamic acid), tetradeca(glutamic acid),        pentadeca(glutamic acid), hexadeca(glutamic acid),        heptadeca(glutamic acid), octadeca(glutamic acid),        nonadeca(glutamic acid), di(aspartic acid), tri(aspartic acid),        tetra(aspartic acid), penta(aspartic acid), hexa(aspartic acid),        hepta(aspartic acid), octa(aspartic acid), nona(aspartic acid),        deca(aspartic acid), undeca(aspartic acid), dodeca(aspartic        acid), trideca(aspartic acid), tetradeca(aspartic acid),        pentadeca(aspartic acid), hexadeca(aspartic acid),        heptadeca(aspartic acid), octadeca(aspartic acid),        nonadeca(aspartic acid), polyethyleneimines, and        polyvinylamines.

In a preferred embodiment, a moiety Hyp^(y) of formula (VI) is selectedfrom the group comprising, in particular consisting of, in bound form,dilysine, trilysine, tetralysine, pentalysine, hexalysine, heptalysine,octalysine, nonalysine, decalysine, undecalysine, dodecalysine,tridecalysine, tetradecalysine, pentadecalysine, hexadecalysine,heptadecalysine, octadecalysine, nonadecalysine, triornithine,tetraornithine, pentaornithine, hexaornithine, heptaornithine,octaornithine, nonaornithine, decaornithine, undecaornithine,dodecaornithine, tridecaornithine, tetradecaornithine,pentadecaornithine, hexadecaornithine, heptadecaornithine,octadecaornithine, nonadecaornithine, tridiaminobutyric acid,tetradiaminobutyric acid, pentadiaminobutyric acid, hexadiaminobutyricacid, heptadiaminobutyric acid, octadiaminobutyric acid,nonadiaminobutyric acid, decadiaminobutyric acid, undecadiaminobutyricacid, dodecadiaminobutyric acid, tridecadiaminobutyric acid,tetradecadiaminobutyric acid, pentadecadiaminobutyric acid,hexadecadiaminobutyric acid, heptadecadiaminobutyric acid,octadecadiaminobutyric acid, nonadecadiaminobutyric acid, di(glutamicacid), tri(glutamic acid), tetra(glutamic acid), penta(glutamic acid),hexa(glutamic acid), hepta(glutamic acid), octa(glutamic acid),nona(glutamic acid), deca(glutamic acid), undeca(glutamic acid),dodeca(glutamic acid), trideca(glutamic acid), tetradeca(glutamic acid),pentadeca(glutamic acid), hexadeca(glutamic acid), heptadeca(glutamicacid), octadeca(glutamic acid), nonadeca(glutamic acid), di(asparticacid), tri(aspartic acid), tetra(aspartic acid), penta(aspartic acid),hexa(aspartic acid), hepta(aspartic acid), octa(aspartic acid),nona(aspartic acid), deca(aspartic acid), undeca(aspartic acid),dodeca(aspartic acid), trideca(aspartic acid), tetradeca(aspartic acid),pentadeca(aspartic acid), hexadeca(aspartic acid), heptadeca(asparticacid), octadeca(aspartic acid), nonadeca(aspartic acid),polyethyleneimines, and low-molecular weight PEI.

More preferably, a moiety Hyp^(y) of formula (VI) is selected from thegroup comprising, more preferably consisting of, in bound form,trilysine, tetralysine, pentalysine, hexalysine, heptalysine,octalysine, nonalysine, decalysine, undecalysine, dodecalysine,tridecalysine, tetradecalysine, pentadecalysine, hexadecalysine, andheptadecalysine, even more preferably a moiety Hyp^(y) of formula (VI)comprises, preferably consists of, in bound form, trilysine, heptalysineor pentadecalysine.

In a preferred embodiment, a moiety Hyp^(y) of formula (VI) has amolecular weight in the range of from 0.1 kDa to 4 kDa, more preferably0.2 kDa to 2 kDa.

In a further preferred embodiment, each branched moiety Hyp^(y) offormula (VI) has at least 1 branching and is conjugated to at least 2moieties X⁰ of formula (I), to at least two moieties Q of formula (II),to at least two moieties Q¹ of either formula (IIaa) or (IIab) or to atleast two moieties X₁ of formula (IIac), (IIad), or (IIb), or to therest of the molecule of formula (IIba), respectively, and has at most 63branchings and is at most conjugated to 64 moieties X⁰ of formula (I),to at least 64 moieties Q of formula (II), to at least 64 moieties Q¹ ofeither formula (IIaa) or (IIab) or to at least 64 moieties X₁ of formula(IIac), (IIad), or (IIb), or to the rest of the molecule of formula(IIba), respectively, more preferably each branched moiety Hyp^(y) offormula (VI) has at least 1 branching and is conjugated to at least 2moieties X⁰ of formula (I), to at least two moieties Q of formula (II),to at least two moieties Q¹ of either formula (IIaa) or (IIab) or to atleast two moieties X₁ of formula (IIac), (IIad), or (IIb), or to therest of the molecule of formula (IIba), respectively, and has at most 31branchings and is at most conjugated to 32 moieties X⁰ of formula (I),to at least 32 moieties Q of formula (II), to at least 32 moieties Q¹ ofeither formula (IIaa) or (IIab) or to at least 32 moieties X¹ of formula(IIac), (IIad), or (IIb), or to the rest of the molecule of formula(IIba), respectively.

In a preferred embodiment, Z¹ of formula (VI) comprises a quaternarycarbon, in particular a quaternary carbon of a branching core moiety B,wherein B of formula (VI) is pentarythritol in bound form. Preferably,each A of formula (VI) is independently a PEG-based polymeric chainterminally attached to the quaternary carbon of pentaerythritol via the—CH₂—O— moieties of the branching core moiety pentaerythritol by apermanent covalent linkage, and the distal end of the PEG-basedpolymeric chain is covalently bound to a branched moiety Hyp^(y) offormula (VI), each branched moiety Hyp^(y) of formula (VI) is conjugatedto the moieties X⁰ of formula (I), to the moieties Q of formula (II), tothe moieties Q¹ of either formula (IIaa) or (IIab) or to the moieties X₁of formula (IIac), (IIad), or (IIb) or to the rest of the molecule offormula (IIba), respectively.

In one preferred embodiment, a branched moiety Hyp^(y) of formula (VI)comprises, preferably consists of branched polyamines comprising atleast 2 amine groups. Preferably, the branched polyamine comprising atleast 2 amine groups, comprises one or more lysine residues in boundform. Preferably, each branched moiety Hyp^(y) of formula (VI) has amolecular weight in the range of from 0.1 kDa to 4 kDa, particular 0.2to 2 kDa. In a preferred embodiment, a moiety B (A-Hyp^(y))_(n) offormula (VI), wherein n=4, consist of the same or different branchedmoieties Hyp^(y) and that each moiety Hyp^(y) can be chosenindependently. In a preferred embodiment, all moieties Hyp_(y) offormula (VI) are the same.

In a preferred embodiment, a moiety Hyp^(y) of formula (VI) comprises,in particular consists of, between 1 and 32 lysines in bound form,preferably of 1, 3, 7 or 15 lysines in bound form, more preferably of 1,3 or 7 lysines in bound form. Most preferably, Hyp^(y) of formula (VI)comprises, in particular consists of heptalysinyl.

Preferably, the moiety B (A-Hyp^(y))_(n) of formula (VI), wherein n ispreferably 4, has a molecular weight in the range of from 1 kDa to 160kDa, more preferably 1 kDa to 80 kDa and even more preferably 10 kDa to40 kDa.

Preferred moieties BA-Hyp^(y))₄ of formula (VI) are selected fromstructures (i-y) to (iii-y):

-   -   wherein    -   dashed lines indicate attachment to a moiety X⁰ of formula (I),        to a moiety Q of formula (II), to a moiety Q¹ of formula (IIaa)        or (IIab) or to a moiety X₁ of formula (IIac), (IIad) or (IIb),        or to the rest of the molecule of formula (IIba), respectively,        provided that one of m1, m2 of formula (I) is 1 and wherein the        carrier is covalently attached to T in case m1, m2=0,    -   p is an integer of from 5 to 2000, preferably from 10 to 1000,        more preferably from 10 to 500, most preferably from 100 to        1000,    -   q is 1 or 2.

In a preferred embodiment, B of formula (VI) is pentaerythritol.

In another preferred embodiment, Z¹ of formula (I), (II), (IIaa),(IIab), (IIac), (IIad), (IIb), or (IIba) is a protein carrier whichcomprises, in particular consists of an amino acid sequence of at least100 amino acid residues.

In another preferred embodiment, the protein carrier Z¹ of formula (I),(II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) is in random coilconformation.

In another preferred embodiment, the protein carrier Z¹ of formula (I),(II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) comprises, inparticular consists of alanine, serine and proline residues.

In the preferred embodiment, the protein carrier Z¹ of formula (I),(II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) comprises, inparticular consists of an amino acid sequence of at least 100 amino acidresidues, and

-   -   wherein the amino acid sequence of at least 100 amino acid        residues is in random coil conformation, and,    -   wherein the amino acid sequence of at least 100 amino acid        residues comprises alanine, serine and proline residues.

Preferably, the protein carrier a protein carrier Z¹ of formula (I),(II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) is composed of anamino acid sequence comprising at least about 100 amino acid residues,at least 100 amino acid residues, consisting of alanine, serine andproline residues which have a random coil conformation at physiologicalconditions. It is understood that the protein carrier Z¹ of formula (I),(II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) may transiently ortemporarily not form a random coil, for example when present in alyophilisate or dried composition.

In one embodiment the protein carrier Z¹ of formula (I), (II), (IIaa),(IIab), (IIac), (IIad), (IIb) or (IIba) has a random coil conformationwith an amino acid sequence of maximally about 3000 amino acid residues,preferably of maximally about 1500 amino acid residues, more preferablyof maximally about 900 amino acid residues, even more preferably ofmaximally about 700 amino acid residues, particularly preferably ofmaximally about 600 amino acid residues. Thus, the amino acid sequenceforming random coil conformation is maximally about 500 amino acidresidues or of maximally about 450 amino acid residues in length.

Accordingly, the protein carrier Z¹ of formula (I), (II), (IIaa),(IIab), (IIac), (IIad), (IIb) or (IIba) in particular the amino acidsequence forming random coil conformation of the protein carrier Z¹ offormula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) isabout 100 to about 3000 amino acid residues in length.

In particular embodiments said amino acid sequence forming random coilconformation of about 100 to 1000 amino acid residues is ascharacterized herein, i.e. comprising alanine, serine and proline asmain or unique residues as defined below.

The protein carrier moiety Z¹ of formula (I), (II), (IIaa), (IIab),(IIac), (IIad), (IIb) or (IIba) consists mainly of the three amino acidresidues alanine, serine and proline, and wherein all three amino acidsare present in a protein carrier moiety Z¹ of formula (I), (II), (IIaa),(IIab), (IIac) (IIad), (IIb) or (IIba), whereby proline residuesrepresent preferably about 4% to about 40% of the protein carrier Z¹ offormula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb), and (IIba).The alanine and serine residues preferably comprise the remaining atleast 60% to 96% of the protein carrier Z¹ of formula (I), (II), (IIaa),(IIab), (IIac) or (IIad). However, as will be detailed herein below saidprotein carrier Z¹ of formula (I), (II), (IIaa), (IIab), or (IIac) mayalso comprise further amino acids differing from alanine, serine, andproline, i.e. as minor constituents.

The term “minor constituent” as used herein means that maximally 10%(i.e. maximally 10 of 100 amino acids) may be different from alanine,serine and proline, preferably maximally 8% (i.e. maximally 8 of 100amino acids) may be different than alanine, serine and proline, morepreferably maximally 6% (i.e. maximally 6 of 100 amino acids) may bedifferent from alanine, serine and proline, even more preferablymaximally 5% (i.e. maximally 5 of 100 amino acids) may be different fromalanine, serine and proline, particularly preferably maximally 4% (i.e.maximally 4 of 100 amino acids) may be different from alanine, serineand proline, more particularly preferably maximally 3% (i.e. maximally 3of 100 amino acids) may be different from alanine, serine and proline,even more particularly preferably maximally 2% (i.e. maximally 2 of 100amino acids) may be different from alanine, serine and proline and mostpreferably maximally 1% (i.e. maximally 1 of 100 of the amino acids)that encode the protein carrier Z¹ of formula (I), (II), (IIaa), (IIab),(IIac) or (IIad) may be different from alanine, serine and proline. Saidamino acids different from alanine, serine and proline may be selectedfrom the group of natural or proteinogenic amino-acids consisting ofArg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Thr,Trp, Tyr, and Val. Minor constituents may also be selected fromnon-naturally occurring amino acids, such as, for example,hydroxyproline or selenomethionine or other modified natural aminoacids.

The term “at least about 100/150/200/250/300/300/350 (etc) amino acidresidues” is not limited to the concise number of amino acid residuesbut also comprises amino acid stretches that comprise an additional 10%to 20% or comprise 10% to 20% less residues. For example “at least about100 amino acid residues” may also comprise 80 to 100 and about 100 to120 amino acid residues.

In one embodiment, the protein carrier Z¹ of formula (I), (II), (IIaa),(IIab), (IIac) or (IIad) comprises a plurality of polymer cassetteswherein said polymer cassettes consist of Ala, Ser, and/or Pro, andwherein no more than 6 consecutive amino acid residues of the polymercassettes, preferably of the protein carrier Z¹ of formula (I), (II),(IIaa), (IIab), (IIac) or (IIad) are identical and wherein said prolineresidues constitute more than 4% and less than 40% of the amino acids ofsaid protein carrier Z¹ of formula (I), (II), (IIaa), (IIab), (IIac) or(IIad).

In one embodiment, the protein carrier moiety Z¹ of formula (I), (II),(IIaa), (IIab), or (IIIac) comprises, preferably consists of a pluralityof amino acid repeats,

-   -   wherein said repeats consist of Ala, Ser, and Pro residues,    -   and wherein no more than 6 consecutive amino acid residues of        the carrier moiety Z¹ of formula (I), (II), (IIaa), (IIab),        (IIac), or (IIad), (IIb) or (IIba) are identical.

In a preferred embodiment, said proline residues constitute more than 4%and less than 40% of the amino acids of the protein carrier moiety Z¹ offormula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba).

In a further preferred embodiment, the protein carrier moiety Z¹ offormula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb), or (IIba)comprises, in particular consists of an amino acid sequence of about 100to 3000 amino acid residues forming random coil conformation.

The protein carrier Z¹ of formula (I), (II), (IIaa), (IIab), (IIac),(IIad), (IIb) or (IIba) may comprise a plurality of identical polymercassettes or a plurality of non-identical polymer cassettes.Non-limiting examples of polymer cassettes consisting of Ala, Ser and/orPro residues are provided herein below; see SEQ ID NO:9, SEQ ID NO:10,SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14 or peptidefragments or multimers of these sequences. A polymer cassette mayconsist of at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or more amino acidresidues, wherein each polymer cassette comprises (an) Ala, Ser, and/orPro residue(s), preferably (an) Ala, Ser, and Pro residue(s).

In one embodiment, the polymer cassette does not comprise more than 100amino acid residues. Preferably, a polymer cassette as defined hereincomprises more than about 4%, preferably more than about 5%, even morepreferably more than about 6%, particularly preferably more than about8%, more particularly preferably more than about 10%, even moreparticularly preferably more than about 15% and most preferably morethan about 20% proline residues. Such polymer cassette as defined hereinpreferably comprises less than about 40% or less than about 35% prolineresidues.

In one embodiment the protein carrier Z¹ of formula (I), (II), (IIaa),(IIab), (IIac), (IIad), (IIb) or (IIba) is of formula (b):

Ser_(x)[Ala_(y)Ser_(z)]_(v)  (b),

-   -   which formula further comprises proline residues as defined        herein and wherein    -   x is independently selected from integer 0 to 6,    -   each y is independently selected from integer ranging of from 1        to 6,    -   each z is independently selected from integer ranging of from 1        to 6.    -   v is any integer so that the protein carrier Z¹ of formula (I),        (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) consists        of at least about 100 amino acid residues, and in particular of        at least about 100 to about 3000 amino acid residues, preferably        to about 2000 and more preferably to about 1000 amino acid        residues.

In one embodiment, all y of formula (b) and z of formula (b) of the vAla_(y) Ser_(z) monomer moieties of formula (b) are identical. Inanother embodiment, the y of formula (b) and z of formula (b) of the vAla_(y) Ser_(z) monomer moieties of formula (b) are different.

In preferred embodiments, the protein carrier Z¹ of formula (I), (II),(IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) comprises no more than 5identical consecutive amino acid residues, more preferably no more than4 identical consecutive amino acid residues and most preferably no morethan 3 identical consecutive amino acid residues.

As already indicated herein above, the protein carrier Z¹ of formula(I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) comprisesproline residues, wherein said proline residues constitute more thanabout 4%, preferably more than about 5%, even more preferably more thanabout 6%, particularly preferably more than about 8%, more particularlypreferably more than about 10%, even more particularly preferably morethan about 15% and most preferably more than about 20% of the aminoacids constituting the protein carrier Z¹ of formula (I), (II), (IIaa),(IIab), (IIac), (IIad), (IIb) or (IIba). Such proline residues may beintroduced at any position in formula (b). Preferably, the prolineresidues may be present in one or more of the v Ala_(y) Ser_(z) monomersof formula (b), and they may be present at the same or at differentpositions.

In another preferred embodiment, the protein carrier Z¹ of formula (I),(II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) comprises morethan about 4% but less than about 50%, preferably more than about 10%but less than about 50% and most preferably more than about 20% but lessthan about 50% alanine residues of the amino acids constituting theprotein carrier Z¹ of formula (I), (II), (IIaa), (IIab), (IIac), (IIad),(IIb) or (IIba).

In a further preferred embodiment, the protein carrier Z¹ of formula(I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) comprisesmore than about 4% and less than about 50%, preferably more than about10% but less than about 50% and most preferably more than about 20% butless than about 50% serine residues of the amino acids constituting theprotein carrier Z¹ of formula (I), (II), (IIaa), (IIab), (IIac), (IIad),(IIb) or (IIba).

Accordingly, the protein carrier Z¹ of formula (I), (II), (IIaa),(IIab), (IIac), (IIad), (IIb) or (IIba) comprises about 35% prolineresidues, about 50% alanine residues and about 15% serine residues ofthe amino acids constituting the protein carrier Z¹ of formula (I),(II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba). Alternatively,the protein carrier Z¹ of formula (I), (II), (IIaa), (IIab), (IIac),(IIad), (IIb) or (IIba) may comprise about 35% proline residues, about15% alanine residues and about 50% serine residues of the amino acidsconstituting the protein carrier Z¹ of formula (I), (II), (IIaa),(IIab), (IIac), (IIad), (IIb) or (IIba).

Preferably, the protein carrier Z¹ of formula (I), (II), (IIaa), (IIab),(IIac), (IIad), (IIb) or (IIba) is comprises one or more of thefollowing alanine-serine polymer cassettes:

SEQ ID NO: 1 AAAASSASSASSSSSAAASA SEQ ID NO: 2 AASAAASSAAASAAAASASSSEQ ID NO: 3 ASASASASASASSAASAASA SEQ ID NO: 4 SAASSSASSSSAASSASAAASEQ ID NO: 5 SSSSAASAASAAAAASSSAS SEQ ID NO: 6 SSASSSAASSSASSSSASAASEQ ID NO: 7 SASASASASASAASSASSAS SEQ ID NO: 8 ASSAAASAAAASSAASASSS

provided that the protein carrier Z¹ of formula (I), (II), (IIaa),(IIab), (IIac), (IIad), (IIb) or (IIba) further comprises prolineresidues as described herein.

The multimers of these alanine-serine polymer cassettes may form randomcoil conformation in case the resulting amino acid sequence furthercomprises proline residues as defined herein above.

In a preferred embodiment, the protein carrier Z¹ of formula (I), (II),(IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) comprises, preferablyconsists of one or more of the following polymer cassettes:

SEQ ID NO: 9 ASPAAPAPASPAAPAPSAPA SEQ ID NO: 10 AAPASPAPAAPSAPAPAAPSSEQ ID No: 11 APSSPSPSAPSSPSPASPSS SEQ ID NO: 15 SAPSSPSPSAPSSPSPASPS

SEQ ID NO:15 corresponds to the herein provided SEQ ID No:11 in acircularly permuted form, wherein the last serine was removed andanother serine was appended as starting amino acid. As a consequence,multimers of this modified sequence possess essentially the sameinternal repeating unit as multimers of the non-modified sequence,except for the very first and the very last residue. Accordingly, SEQ IDNO:15 may be considered as an example of a further polymer cassette forthe protein carrier Z¹ of formula (I), (II), (IIaa), (IIab), (IIac),(IIad), (IIb) or (IIba). It is clear for the person skilled in the artthat also other polymer cassettes and (shorter) peptide fragments orcircularly permuted versions of the herein provided amino acid polymersmay be used as polymer cassettes for the protein carrier Z¹ of formula(I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba).

Yet, even further and illustrative amino acid polymers forming randomcoil conformation may comprise amino acid sequences that may be selectedfrom the group consisting of:

SEQ ID NO: 12 SSPSAPSPSSPASPSPSSPA, SEQ ID NO: 13AASPAAPSAPPAAASPAAPSAPPA, and SEQ ID NO: 14 ASAAAPAAASAAASAPSAAA.

Therefore, preferred polymer cassettes for Z¹ of formula (I), (II),(IIaa), (IIab), (IIac), (Had), (IIb) or (IIba) are selected from thefollowing sequences:

(SEQ ID NO: 9) ASPAAPAPASPAAPAPSAPA, (SEQ ID NO: 10)AAPASPAPAAPSAPAPAAPS, (SEQ ID NO: 11) APSSPSPSAPSSPSPASPSS,(SEQ ID NO: 12) SSPSAPSPSSPASPSPSSPA, (SEQ ID NO: 13)AASPAAPSAPPAAASPAAPSAPPA, and (SEQ ID NO: 14) ASAAAPAAASAAASAPSAAA;

-   -   or circular permuted versions or (a) multimer(s) of these        sequences as a whole or parts of these sequences.

In one embodiment, the protein carrier moiety Z¹ of formula (I), (II),(IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) comprises at least oneamino acid sequence selected from the group consisting of:

(SEQ ID NO: 9) ASPAAPAPASPAAPAPSAPA, (SEQ ID NO: 10)AAPASPAPAAPSAPAPAAPS, (SEQ ID NO: 11) APSSPSPSAPSSPSPASPSS,(SEQ ID NO: 12) SSPSAPSPSSPASPSPSSPA, (SEQ ID NO: 13)AASPAAPSAPPAAASPAAPSAPPA, and (SEQ ID NO: 14) ASAAAPAAASAAASAPSAAA;

and circular permuted versions or (a) multimer(s) of these sequences asa whole or parts of these sequences.

Again, also (a) peptide fragment(s) or (a) multimer(s) or circularlypermuted versions of these sequences and the sequences provided hereinabove may be employed as polymer cassettes for the protein carrier Z¹ offormula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba).

Accordingly, the exemplified polymer cassettes may also provide forindividual peptide fragments which may be newly combined to form furtherpolymer cassettes.

In accordance with the above, the protein carrier Z¹ of formula (I),(II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) may comprise amultimer consisting of either one of the amino acid sequences with SEQID NO:9, 10, 11, 12, 13 or 14 as disclosed herein above or may comprisea multimer consisting of more than one of amino acid sequences SEQ IDNO:9, 10, 11, 12, 13 and 14. Furthermore, it is envisaged that alsopeptide fragments or circularly permuted versions of these exemplifiedsequences may be used to build up further polymer cassettes of theprotein carrier Z¹ of formula (I), (II), (IIaa), (IIab), (IIac), (IIad),(IIb) or (IIba).

In another embodiment, the protein carrier Z¹ of formula (I), (II),(IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) may comprise a multimercomprising, preferably consisting of a (circular) permutation of theamino acid sequence selected from the group consisting of SEQ ID NOs:9,10, 11, 12, 13, 14, 15 and (a) multimers(s) of these (circular)permutated sequences.

In yet another embodiment, the protein carrier Z¹ of formula (I), (II),(IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) may comprise, preferablyconsist of a multimer consisting of a peptide fragment/part of the aminoacid sequence selected from the group consisting of SEQ ID NO: 9, 10,12, 13, 14, 15 and (a) multimers(s) of these exemplified polymercassettes.

Peptide fragments of these sequences to be employed for the generationof the protein carrier Z¹ of formula (I), (II), (IIaa), (IIab), (IIac),(IIad), (IIb) or (IIba) may consist of at least 3, preferably of atleast 4, more preferably of at least 5, even more preferably of at least6, still more preferably of at least 8, particularly preferably of atleast 10, more particularly preferably of at least 12, even moreparticularly preferably of at least 14, preferably of at least 6, stillmore preferably of at least 8, particularly preferably of at least 10,more particularly preferably of at least 12, even more particularlypreferably of at least 14, even more particularly preferably of at least16, and most preferably of at least 18 consecutive amino acids of theamino acid sequence selected from the group consisting of said SEQ IDNOs: 9, 10, 11, 12, 13 and 14.

For example, individual peptide fragments of the polymer cassettes maybe combined to further individual polymer cassettes as long as theabove-identified rules for the overall distribution and amount ofalanine, serine and proline are respected. Again, these polymercassettes may also comprise further amino acid residues, however only asminimal or minor constituents, i. e. maximally 10%, preferably maximally2% of the individual polymer cassette. Said individual polymer cassettesconsist of at least about 100 amino acid residues. Individual polymercassettes may be combined in order to form longer random coil formingamino acid polymers, whereby a maximal length of the protein carrier Z¹of formula (I), (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) isabout 3000 amino acids. A preferred minor constituent of the proteincarrier Z¹, (II), (IIaa), (IIab), (IIac), (IIad), (IIb) or (IIba) islysine.

In another embodiment the carrier Z¹ is a C₁₀₋₂₄ fatty acid, i.e. acarboxylic acid with a linear carbon chain having 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 carbon atoms, which is eithersaturated or partially or fully unsaturated.

In another embodiment the carrier Z¹ has the structure of formula (VII):

BA)_(n)  (VII),

wherein

B is branching core,

A is a poly(ethylene glycol)-based polymeric chain, and

n is an integer of from 3 to 32.

In a preferred embodiment, the branching core B of formula (VII)comprises, preferably consists of a moiety selected from:

-   -   a polyalcohol comprising at least 2 hydroxyl groups (preferably        further comprising a functional group, which is preferably an        additional amino group or a carboxylic acid group, more        preferably an additional carboxylic acid group),    -   preferably B is selected from glycerol, pentaerythritol,        dipentaerythritol, tripentaerythritol, hexaglycerine, sucrose,        sorbitol, fructose, mannitol, glucose, cellulose, amyloses,        starches, hydroxyalkyl starches, polyvinylalcohols, dextranes,        and hyualuronans, erythritol, threitol, arabitol, xylitol,        ribitol, dulcitol, iditol; more preferably from glycerol,        pentaerythritol, dipentaerythritol, tripentaerythritol,        hexaglycerine, sucrose, sorbitol, fructose, mannitol, glucose,        cellulose, amyloses, starches, hydroxyalkyl starches,        polyvinylalcohols, dextranes, and hyualuronans.    -   or a polyamine comprising at least 2 amine groups (preferably        further comprising a functional group, which is preferably an        additional hydroxyl group or a carboxylic acid group, more        preferably a carboxylic acid group),    -   preferably selected from ornithine, diornithine, triornithine,        tetraornithine, pentaornithine, hexaornithine, heptaornithine,        octaornithine, nonaornithine, decaornithine, undecaornithine,        dodecaornithine, tridecaornithine, tetradecaornithine,        pentadecaornithine, hexadecaornithine, heptadecaornithine,        octadecaornithine, nonadecaornithine, diaminobutyric acid,        di(diaminobutyric acid), tri(diaminobutyric acid),        tetra(diaminobutyric acid), penta(diaminobutyric acid),        hexa(diaminobutyric acid), hepta(diaminobutyric acid),        octa(diaminobutyric acid), nona(diaminobutyric acid),        deca(diaminobutyric acid), undeca(diaminobutyric acid),        dodeca(diaminobutyric acid), trideca(diaminobutyric acid),        tetradeca(diaminobutyric acid), pentadeca(diaminobutyric acid),        hexadeca(diaminobutyric acid), heptadeca(diaminobutyric acid),        octadeca(diaminobutyric acid), nonadeca(diaminobutyric acid),        lysine, dilysine, trilysine, tetralysine, pentalysine,        hexalysine, heptalysine, octalysine, nonalysine, decalysine,        undecalysine, dodecalysine, tridecalysine, tetradecalysine,        pentadecalysine, hexadecalysine, heptadecalysine,        octadecalysine, nonadecalysine, oligolysines,        polyethyleneimines, and polyvinylamines;    -   wherein the polyalcohol or polyamine is in bound form.

In a preferred embodiment, the branching core B of formula (VII)comprises, preferably consists of pentaerithritol.

Preferably, a poly(ethylene glycol)-based polymeric chain A connected tothe branching core B of formula (VII) consists of a linear PEG chain, ofwhich one terminus is connected to B of formula (VII) and the otherterminus is connected to X⁰ of formula (I), to Q (if the carrier-linkedtreprostinil prodrug is of formula (II)), to Q¹ (if the carrier-linkedtreprostinil prodrug is of formula (IIaa) or (IIab)) or to X₁ (if thecarrier-linked treprostinil prodrug is of formula (IIac), (IIad), or(IIb)), or to the rest of the molecule (if the carrier-linkedtreprostinil prodrug is of formula (IIba)), respectively.

It is understood that a PEG-based chain A of formula (VII) mayoptionally be terminated in case of a branched PEG chain and/or mayoptionally be interrupted in case of a branched or linear PEG chain byalkyl or aryl groups and may optionally be substituted with heteroatomsand/or functional groups.

Preferably, the carrier Z¹ of formula (VII) is a multi-arm PEGderivative as, for instance, detailed in the products list of JenKemTechnology, USA (accessed by download fromhttp://jenkemusa.net/pegproducts2.aspx on Mar. 8, 2011), such as a4-arm-PEG derivative, in particular comprising a pentaerythritol core,an 8-arm-PEG derivative comprising a hexaglycerin core, and an 8-arm-PEGderivative comprising a tripentaerythritol core. Most preferred aresub-structures B (A)_(n) of formula (VI) comprising, in particularconsisting of, moieties selected from:

a 4-arm PEG Amine comprising a pentaerythritol core:

with n ranging from 20 to 500;

a 4-arm PEG Carboxyl comprising a pentaerythritol core:

with n ranging from 20 to 500;

an 8-arm PEG Amine comprising a hexaglycerin core:

with n ranging from 20 to 500; and

R=hexaglycerin core structure;

an 8-arm PEG Carboxyl comprising a hexaglycerin core:

with n ranging from 20 to 500; and

R=hexaglycerin core structure;

an 8-arm PEG Amine comprising a tripentaerythritol core:

with n ranging from 20 to 500;

and R=tripentaerythritol core structure;

an 8-arm PEG Carboxyl comprising a tripentaerythritol core:

with n ranging from 20 to 500; and

R=tripentaerythritol core structure;

a 6-arm PEG Amine comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500; and

R=sorbitol or dipentaerythritol;

a 6-arm PEG Carboxyl comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500; and

R=sorbitol or dipentaerythritol;

a 6-arm PEG Amine comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500;

and R=sorbitol or dipentaerythritol;

a 6-arm PEG Carboxyl comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500; and

R=sorbitol or dipentaerythritol;

a 4-arm PEG Amine comprising a pentaerythritol core:

with n ranging from 20 to 500;

a 4-arm PEG Carboxyl comprising a pentaerythritol core:

with n ranging from 20 to 500;

an 8-arm PEG Amine comprising a hexaglycerin core:

with n ranging from 20 to 500; and

R=hexaglycerin core structure;

an 8-arm PEG Carboxyl comprising a hexaglycerin core:

with n ranging from 20 to 500; and

R=hexaglycerin core structure;

an 8-arm PEG Amine comprising a tripentaerythritol core:

with n ranging from 20 to 500;

and R=tripentaerythritol core structure;

and an 8-arm PEG Carboxyl comprising a tripentaerythritol core:

with n ranging from 20 to 500; and

R=tripentaerythritol core structure;

a 6-arm PEG Amine comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500; and

R=comprising a sorbitol or dipentaerythritol core;

a 6-arm PEG Carboxyl comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500; and

R=comprising a sorbitol or dipentaerythritol core;

a 6-arm PEG Amine comprising a sorbitol or dipentaerythritol core:

with n ranging from 20 to 500;

and R=comprising a sorbitol or dipentaerythritol core;

and a 6-arm PEG Carboxyl comprising a sorbitol or dipentaerythritolcore:

with n ranging from 20 to 500; and

R=comprising a sorbitol or dipentaerythritol core;

wherein dashed lines indicate attachment to X⁰ of formula (I), to Q (ifthe carrier-linked treprostinil prodrug is of formula (II)), to Q¹ (ifthe carrier-linked treprostinil prodrug is of formula (IIaa) or (IIab))or to X₁ (if the carrier-linked treprostinil prodrug is of formula(IIac), (IIad), or (IIb)), or to the rest of the molecule (if thecarrier-linked treprostinil prodrug is of formula (IIba)), respectively.

In a preferred embodiment, the molecular weight of the carrier B-(A)_(n)of formula (VII) ranges from 1 kDa to 80 kDa, more preferably 1 kDa to40 kDa and even more preferably 10 kDa to 40 kDa.

More preferably, the carrier of formula (VII) has the structure offormula (VIIa):

wherein

t ranges from 80 to 160;

w ranges from 2 to 6;

and dashed lines indicate attachment to the rest of the carrier-linkedtreprostinil prodrug, i.e. a moiety X⁰ of formula (I), to a moiety Q (ifthe carrier-linked treprostinil prodrug is of formula (II)), to a moietyQ¹ (if the carrier-linked treprostinil prodrug is of formula (IIaa) or(IIab)) or to a moiety X₁ (if the carrier-linked treprostinil prodrug isof formula (IIac), (IIad), or (IIb)), or to the rest of the molecule (ifthe carrier-linked treprostinil prodrug is of formula (IIba)),respectively.

Preferably, w is 2 or 3.

Most preferably, the carrier-linked treprostinil prodrug has thestructure of formula (IIc):

wherein y is 4 and Z¹ represents a moiety (IIc):

wherein dashed lines indicate attachment to the rest of the structure offormula (IIc), t ranges from 80 to 160 and w is 2 or 3.

In another preferred embodiment, the present invention relates to apharmaceutical composition comprising a carrier-linked treprostinilprodrug, wherein said pharmaceutical composition is characterized inthat the prostacyclin compound releases prostacyclin in aplasma-independent manner. Preferably, the prostacyclin compoundreleases prostacyclin in an enzyme-independent manner.

The term “plasma-independent” means that the release kinetics ofprostacyclin from the prostacyclin compound measured at 37° C.independently in buffer at pH 7.4 and in 80% buffered plasma at pH 7.4varies by no more than 50%, preferably by no more than 40%, morepreferably by no more than 30%, even more preferably by no more than 20%and most preferably by no more than 10%.

The term “enzyme-independent” means that the release of prostacyclinfrom the prostacyclin compound does not require the presence of enzymes.

Another aspect of the present invention is a pharmaceutical compositioncomprising a carrier-linked treprostinil prodrug or a pharmaceuticallyacceptable salt of the present invention, optionally together with oneor more pharmaceutically acceptable excipients.

The pharmaceutical composition is further described in the followingparagraphs.

The pharmaceutical composition comprising the carrier-linkedtreprostinil prodrug of the present invention may be provided as aliquid composition or as a dry composition. Suitable methods of dryingare, for example, spray-drying and lyophilization (freeze-drying). Apreferred method of drying is lyophilization.

Preferably, the carrier-linked treprostinil prodrug is sufficientlydosed in the composition to provide a therapeutically effective amountof treprostinil for at least 12 hours in one application. Morepreferably, one application of the pharmaceutical composition comprisingthe carrier-linked treprostinil prodrug is sufficient for at least 1day, such as two days, three days, four days, five days, six days, orseven days, such as two weeks, three weeks or four weeks.

In one embodiment, the present invention relates to a pharmaceuticalcomposition

-   -   (i) wherein the carrier-linked treprostinil prodrug of the        present invention is sufficiently dosed in the pharmaceutical        composition to provide a therapeutically effective amount of        treprostinil for at least 12 hours in one application, and/or    -   (ii) wherein a single dose of the pharmaceutical composition        comprises about 2 to about 6, preferably about 4 mg        treprostinil.

In a preferred embodiment, the a single dose of a liquid pharmaceuticalcomposition of the present invention has a volume of about 0.1 to about10 ml, preferably about 0.5 to about 5 ml, even more preferably about0.5 to about 2 ml, in particular about 1 ml.

“About” according to the present invention is understood as meaning theexperimental error range, in particular ±5% or ±10%.

The pharmaceutical composition of carrier-linked treprostinil prodrugaccording to the present invention optionally comprises one or moreexcipients.

Excipients may be categorized as buffering agents, isotonicitymodifiers, preservatives, stabilizers, anti-adsorption agents, oxidationprotection agents, viscosifiers/viscosity enhancing agents, or otherauxiliary agents. In some cases, these ingredients may have dual ortriple functions. The pharmaceutical compositions of carrier-linkedtreprostinil prodrugs according to the present invention contain one ormore excipients, selected from the groups consisting of:

-   (i) Buffering agents: physiologically tolerated buffers to maintain    pH in a desired range, such as sodium phosphate, bicarbonate,    succinate, histidine, citrate and acetate, sulphate, nitrate,    chloride, pyruvate. Antacids such as Mg(OH)₂ or ZnCO₃ may be also    used. Buffering capacity may be adjusted to match the conditions    most sensitive to pH stability-   (ii) Isotonicity modifiers: to minimize pain that can result from    cell damage due to osmotic pressure differences at the injection    depot. Glycerin and sodium chloride are examples. Effective    concentrations can be determined by osmometry using an assumed    osmolality of 285-315 mOsmol/kg for serum-   (iii) Preservatives and/or antimicrobials: multidose parenteral    preparations require the addition of preservatives at a sufficient    concentration to minimize risk of patients becoming infected upon    injection and corresponding regulatory requirements have been    established. Typical preservatives include m-cresol, phenol,    methylparaben, ethylparaben, propylparaben, butylparaben,    chlorobutanol, benzyl alcohol, phenylmercuric nitrate, thimerosol,    sorbic acid, potassium sorbate, benzoic acid, chlorocresol, and    benzalkonium chloride-   (iv) Stabilizers: Stabilization is achieved by strengthening of the    protein-stabilizing forces, by destabilization of the denatured    state, or by direct binding of excipients to the protein.    Stabilizers may be amino acids such as alanine, arginine, aspartic    acid, glycine, histidine, lysine, proline, sugars such as glucose,    sucrose, trehalose, polyols such as glycerol, mannitol, sorbitol,    salts such as potassium phosphate, sodium sulphate, chelating agents    such as EDTA, hexaphosphate, ligands such as divalent metal ions    (zinc, calcium, etc.), other salts or organic molecules such as    phenolic derivatives. In addition, oligomers or polymers such as    cyclodextrins, dextran, dendrimers, PEG or PVP or protamine or HSA    may be used-   (v) Anti-adsorption agents: Mainly ionic or non-ionic surfactants or    other proteins or soluble polymers are used to coat or adsorb    competitively to the inner surface of the composition's or    composition's container. Suitable surfactants are e.g., alkyl    sulfates, such as ammonium lauryl sulfate and sodium lauryl sulfate;    alkyl ether sulfates, such as sodium laureth sulfate and sodium    myreth sulfate; sulfonates such as dioctyl sodium sulfosuccinates,    perfluorooctanesulfonates, perfluorobutanesulfonates, alkyl benzene    sulfonates; phosphates, such as alkyl aryl ether phosphates and    alkyl ether phosphates; carboxylates, such as fatty acid salts    (soaps) or sodium stearate, sodium lauroyl sarcosinate,    perfluorononanoate, perfluorooctanoate; octenidine dihydrochloride;    quaternary ammonium cations such as cetyl trimethylammonium bromide,    cetyl trimethylammonium chloride, cetylpyridinium chloride,    polyethoxylated tallow amine, benzalkonium chloride, benzethonium    chloride, 5-bromo-5-nitor-1,3-dioxane, dimethyldioctadecylammonium    chloride, dioctadecyldimethylammonium bromide; zwitterionics, such    as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate,    cocamidopropyl hydroxysultaine, amino acids, imino acids,    cocamidopropyl betaine, lecithin; fatty alcohols, such as cetyl    alcohol, stearyl alcohol, cetostearyl alcohol, oleyl alcohol;    polyoxyethylene glycol alkyl ethers, such as octaethylene glycol    monododecyl ether, pentaethylene glycol monododecyl ether;    polyoxypropylene glycol alkyl ethers; glucoside alkyl ethers, such    as decyl glucoside, lauryl glucoside, octyl glucoside;    polyoxyethylene glycol octylphenol ethers such as Triton X-100;    polyoxyethylene glycol alkylphenol ethers such as nonoxynol-9;    glycerol alkyl esters such as glyceryl laurate; polyoxyethylene    glycol sorbitan alkyl esters such as polysorbates; sorbitan alkyl    esters; cocamide MEA and cocamide DEA; dodecyl dimethylamine oxide;    block copolymers of polyethylene glycol and polypropylene glycol,    such as poloxamers (Pluronic F-68), PEG dodecyl ether (Brij 35),    polysorbate 20 and 80; other anti-absorption agents are dextran,    polyethylene glycol, PEG-polyhistidine, BSA and HSA and gelatines.    Chosen concentration and type of excipient depends on the effect to    be avoided but typically a monolayer of surfactant is formed at the    interface just above the CMC value-   (vi) Lyo- and/or cryoprotectants: During freeze- or spray drying,    excipients may counteract the destabilizing effects caused by    hydrogen bond breaking and water removal. For this purpose sugars    and polyols may be used but corresponding positive effects have also    been observed for surfactants, amino acids, non-aqueous solvents,    and other peptides. Trehalose is particularly efficient at reducing    moisture-induced aggregation and also improves thermal stability    potentially caused by exposure of protein hydrophobic groups to    water. Mannitol and sucrose may also be used, either as solely    o/cryoprotectant or in combination with each other where higher    ratios of mannitol:sucrose are known to enhance physical stability    of a lyophilized cake. Mannitol may also be combined with trehalose.    Trehalose may also be combined with sorbitol or sorbitol used as the    sole protectant. Starch or starch derivatives may also be used-   (vii) Oxidation protection agents: antioxidants such as ascorbic    acid, ectoine, methionine, glutathione, monothioglycerol, morin,    polyethylenimine (PEI), propyl gallate, vitamin E, chelating agents    such aus citric acid, EDTA, hexaphosphate, thioglycolic acid-   (viii) Spreading or diffusing agent: modifies the permeability of    connective tissue through the hydrolysis of components of the    extracellular matrix in the intrastitial space such as but not    limited to hyaluronic acid, a polysaccharide found in the    intercellular space of connective tissue. A spreading agent such as    but not limited to hyaluronidase temporarily decreases the viscosity    of the extracellular matrix and promotes diffusion of injected    drugs.-   (ix) Other auxiliary agents: such as wetting agents, viscosity    modifiers, antibiotics, hyaluronidase. Acids and bases such as    hydrochloric acid and sodium hydroxide are auxiliary agents    necessary for pH adjustment during manufacture.

In a general embodiment the pharmaceutical composition comprising thecarrier-linked treprostinil prodrugs of the present invention in eitherdry or liquid form may be provided as a single or multiple dosecomposition.

In one embodiment of the present invention, the liquid or drypharmaceutical composition comprising the carrier-linked treprostinilprodrug is provided as a single dose, meaning that the container inwhich it is supplied contains one pharmaceutical dose.

Alternatively, the liquid or dry pharmaceutical composition comprisingthe carrier-linked treprostinil prodrug is a multiple dose composition,meaning that the container in which it is supplied contains more thanone therapeutic dose, i.e., a multiple dose composition contains atleast 2 doses. Such multiple dose composition of carrier-linkedtreprostinil prodrug can either be used for different patients in needthereof or can be used for one patient, wherein the remaining doses arestored after the application of the first dose until needed.

In another aspect of the present invention the pharmaceuticalcomposition is in a container. Suitable containers for liquid or drycompositions are, for example, syringes, vials, vials with stopper andseal, ampoules, and cartridges. In particular, the liquid or drycomposition comprising the carrier-linked treprostinil prodrug accordingto the present invention is provided in a syringe. If the pharmaceuticalcomposition comprising the carrier-linked treprostinil prodrug is a drypharmaceutical composition the container preferably is a dual-chambersyringe. In such embodiment, said dry pharmaceutical composition isprovided in a first chamber of the dual-chamber syringe andreconstitution solution is provided in the second chamber of thedual-chamber syringe.

Prior to applying the dry composition of carrier-linked treprostinilprodrug to a patient in need thereof, the dry composition isreconstituted. Reconstitution can take place in the container in whichthe dry composition of carrier-linked treprostinil prodrug is provided,such as in a vial, syringe, dual-chamber syringe, ampoule, andcartridge. Reconstitution is done by adding a predefined amount ofreconstitution solution to the dry composition. Reconstitution solutionsare sterile liquids, such as water or buffer, which may contain furtheradditives, such as preservatives and/or antimicrobials, such as, forexample, benzyl alcohol and cresol. Preferably, the reconstitutionsolution is sterile water. When a dry composition is reconstituted, itis referred to as a “reconstituted pharmaceutical composition” or“reconstituted composition”.

An additional aspect of the present invention relates to the method ofadministration of a reconstituted or liquid pharmaceutical compositioncomprising the carrier-linked treprostinil prodrug of the presentinvention. The pharmaceutical composition comprising carrier-linkedtreprostinil prodrug may be administered by methods of inhalation,injection or infusion, including intradermal, subcutaneous,intramuscular, intravenous, intraosseous, and intraperitoneal.Preferably, the pharmaceutical composition comprising carrier-linkedtreprostinil prodrug is administered subcutaneously.

The preferred method of administration for dry pharmaceuticalcompositions comprising the carrier-linked treprostinil prodrugs of thepresent invention is via inhalation.

Therefore, in a preferred embodiment, the present invention relates to acarrier-linked treprostinil prodrug or a pharmaceutically acceptablesalt thereof of the present invention or a pharmaceutical composition ofthe present invention, for use as medicament for topical, enteraladministration, parenteral administration, inhalation, injection, orinfusion, intraarticular, intradermal, subcutaneous, intramuscular,intravenous, intraosseous, and intraperitoneal, intrathecal,intracapsular, intraorbital, intracardiac, transtracheal, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intraventricularor intrasternal administration, preferably for subcutaneousadministration.

In another embodiment, a first carrier-linked treprostinil prodrug ofthe present invention is administered via a first method ofadministration and a second carrier-linked treprostinil prodrug of thepresent invention is administered via a second method of administration,either simultaneously or consecutively. Said first and second method ofadministration can be any combination of topical, enteraladministration, parenteral administration, inhalation, injection, orinfusion, intraarticular, intradermal, subcutaneous, intramuscular,intravenous, intraosseous, and intraperitoneal, intrathecal,intracapsular, intraorbital, intracardiac, transtracheal, subcuticular,intraarticular, subcapsular, subarachnoid, intraspinal, intraventricularor intrasternal administration.

Therefore, in another preferred embodiment, the present inventionrelates to a carrier-linked treprostinil prodrug or a pharmaceuticallyacceptable salt thereof of the present invention or a pharmaceuticalcomposition of the present invention, wherein such water-solublecarrier-linked prodrug or pharmaceutically acceptable salt thereof orpharmaceutical composition is suitable to be administered to a patientvia topical, enteral or parenteral administration and by methods ofexternal application, inhalation, injection or infusion, includingintraarticular, intradermal, subcutaneous, intramuscular, intravenous,intraosseous, and intraperitoneal, intrathecal, intracapsular,intraorbital, intracardiac, transtracheal, subcuticular, intraarticular,subcapsular, subarachnoid, intraspinal, intraventricular andintrasternal application, preferably via subcutaneous administration.

A further aspect is a method of preparing a reconstituted compositioncomprising a therapeutically effective amount of carrier-linkedtreprostinil prodrug of the present invention, and optionally one ormore pharmaceutically acceptable excipients, the method comprising thestep of

-   -   contacting the pharmaceutical composition comprising        carrier-linked treprostinil prodrug of the present invention        with a reconstitution solution.

Another aspect is a reconstituted pharmaceutical composition comprisinga therapeutically effective amount of the carrier-linked treprostinilprodrug of the present invention, and optionally one or morepharmaceutically acceptable excipients.

Another aspect of the present invention is the method of manufacturing adry composition of carrier-linked treprostinil prodrug. In oneembodiment, such dry composition is made by

-   -   (i) admixing the carrier-linked treprostinil prodrug with one or        more excipients,    -   (ii) transferring amounts equivalent to single or multiple doses        into a suitable container,    -   (iii) drying the composition in said container, and    -   (iv) sealing the container.

Suitable containers are vials, syringes, dual-chamber syringes,ampoules, and cartridges.

Another aspect of the present invention is a kit of parts.

If the administration device is simply a hypodermic syringe then the kitmay comprise the syringe, a needle and a container comprising the drypharmaceutical composition of carrier-linked treprostinil prodrug foruse with the syringe and a second container comprising thereconstitution solution.

If the pharmaceutical composition is a liquid composition then the kitmay comprise the syringe, a needle and a container comprising the liquidcomposition of carrier-linked treprostinil prodrug for use with thesyringe.

In more preferred embodiments, the injection device is other than asimple hypodermic syringe and so the separate container withreconstituted or liquid carrier-linked treprostinil prodrug is adaptedto engage with the injection device such that in use the liquidcomposition in the container is in fluid connection with the outlet ofthe injection device. Examples of administration devices include but arenot limited to hypodermic syringes and pen injector devices.Particularly preferred injection devices are the pen injectors in whichcase the container is a cartridge, preferably a disposable cartridge.Optionally, the kit of parts comprises a safety device for the needlewhich can be used to cap or cover the needle after use to preventinjury.

A preferred kit of parts comprises a needle and a container containingthe composition according to the present invention and optionallyfurther containing a reconstitution solution, the container beingadapted for use with the needle. Preferably, the container is adual-chamber syringe.

In another aspect, the invention provides a cartridge comprising apharmaceutical composition of carrier-linked treprostinil prodrug ashereinbefore described for use with a pen injector device. The cartridgemay contain a single dose or multiplicity of doses of the carrier-linkedtreprostinil prodrug.

Yet another aspect of the present invention is a carrier-linkedtreprostinil prodrug of the present invention or a pharmaceuticalcomposition of the present invention for use as a medicament.

In case the carrier-linked treprostinil prodrugs according to theinvention contain one or more acidic or basic groups, the invention alsocomprises their corresponding pharmaceutically or toxicologicallyacceptable salts, in particular their pharmaceutically utilizable salts.Thus, the carrier-linked treprostinil prodrugs according to theinvention which contain acidic groups can be used according to theinvention, for example, as alkali metal salts, alkaline earth metalsalts or as ammonium salts. More precise examples of such salts includesodium salts, potassium salts, calcium salts, magnesium salts or saltswith ammonia or organic amines such as, for example, ethylamine,ethanolamine, triethanolamine or amino acids. Carrier-linkedtreprostinil prodrugs according to the invention which contain one ormore basic groups, i.e. groups which can be protonated, can be presentand can be used according to the invention in the form of their additionsalts with inorganic or organic acids. Examples for suitable acidsinclude hydrogen chloride, hydrogen bromide, phosphoric acid, sulfuricacid, nitric acid, methanesulfonic acid, p-toluenesulfonic acid,naphthalenedisulfonic acids, oxalic acid, acetic acid, tartaric acid,lactic acid, salicylic acid, benzoic acid, formic acid, propionic acid,pivalic acid, diethylacetic acid, malonic acid, succinic acid, pimelicacid, fumaric acid, maleic acid, malic acid, sulfaminic acid,phenylpropionic acid, gluconic acid, ascorbic acid, isonicotinic acid,citric acid, adipic acid, and other acids known to the person skilled inthe art. If the carrier-linked treprostinil prodrugs according to theinvention simultaneously contain acidic and basic groups in themolecule, the invention also includes, in addition to the salt formsmentioned, inner salts or betaines (zwitterions). The respective saltscan be obtained by customary methods which are known to the personskilled in the art like, for example by contacting these with an organicor inorganic acid or base in a solvent or dispersant, or by anionexchange or cation exchange with other salts. The present invention alsoincludes all salts of the prodrugs which, owing to low physiologicalcompatibility, are not directly suitable for use in pharmaceuticals butwhich can be used, for example, as intermediates for chemical reactionsor for the preparation of pharmaceutically acceptable salts.

Yet another aspect of the present invention is a method of treating,controlling, delaying or preventing in a mammalian patient, preferablyin a human, in need of the treatment of one or more conditions, diseasesor disorders comprising administering to said patient a therapeuticallyeffective amount of a carrier-linked treprostinil prodrug of the presentinvention or a pharmaceutical composition comprising the carrier-linkedtreprostinil prodrug of the present invention or a pharmaceuticallyacceptable salt thereof.

Said conditions, diseases or disorders that can be prevented and/ortreated with the carrier-linked treprostinil prodrug of the presentinvention are, for example, pulmonary hypertension, ischemic diseases(e.g. peripheral vascular disease including peripheral arterial disease,Raynaud's phenomenon including Raynaud's disease and Raynaud's syndrome,scleroderma including systemic sclerosis, myocardial ischemia, ischemicstroke, renal insufficiency), ischemic ulcers including digital ulcers,heart failure (including congestive heart failure), portopulmonaryhypertension, interstitial lung disease, idiopathic pulmonary fibrosis,conditions requiring anticoagulation (e.g., post MI, post cardiacsurgery), thrombotic microangiopathy, extracorporeal circulation,central retinal vein occlusion, atherosclerosis, inflammatory diseases(e.g., COPD, psoriasis), hypertension (e.g., preeclampsia), reproductionand parturition, cancer or other conditions of unregulated cell growth,cell/tissue preservation and other emerging therapeutic areas whereprostacyclin treatment appears to have a beneficial role, preferablypulmonary arterial hypertension.

In one embodiment, the present invention relates to a carrier-linkedtreprostinil prodrug or a pharmaceutically acceptable salt thereof or apharmaceutical composition of the present invention for use in a methodof treating or preventing a disease or disorder which can be treatedand/or prevented by treprostinil.

In a preferred embodiment, the disease or disorder is pulmonary arterialhypertension.

In a further embodiment, the present invention relates to the use of acarrier-linked treprostinil prodrug or a pharmaceutically acceptablesalt thereof or a pharmaceutical composition of the present inventionfor the preparation of a medicament for the treatment and/or preventionof a disease or disorder which can be treated and/or prevented bytreprostinil.

In a preferred embodiment, the disease or disorder is pulmonary arterialhypertension.

In one embodiment the carrier-linked treprostinil prodrug of the presentinvention can be administered by different routes of administration tothe same patient, for example but not limited to subcutaneous injectionand inhalation.

In another embodiment the carrier-linked treprostinil prodrug of thepresent invention can be administered in combination with one or moreadditional drug(s) or biologically active moiety/moieties, either intheir free form(s) or as prodrug(s). In another embodiment such one ormore additional drug(s) are administered together with thecarrier-linked treprostinil prodrug of the present invention using thesame or different route(s) of administration. It is preferred that theone or more additional drug(s) and the carrier-linked treprostinilprodrug of the present invention are administered in a fixed dosecombination.

Accordingly, such one or more additional active agents can beadministered separately from the carrier-linked treprostinil prodrug.Particular additional drugs or biologically active moieties that can beadministered in combination with the carrier-linked treprostinil prodrugmay depend on a particular disease or condition for treatment orprevention of which the carrier-linked treprostinil prodrug can beadministered. In some cases, the additional drug/biologically activemoiety can be a cardiovascular agent such as a prostacyclin compound,mediators of NO activity, calcium channel blocker, a phosphodiesteraseinhibitor, diuretics, an endothelial antagonist, or an antiplateletagent.

In another object of the invention, the carrier-linked treprostinilprodrug may be administered in combination with an inhaled prostacyclin.

Another subject of the present invention is a method for the synthesisof a carrier-linked treprostinil prodrug or a pharmaceuticallyacceptable salt thereof of the present invention. Carrier-linkedtreprostinil prodrugs or precursors of such prodrugs according to thepresent invention may be prepared by known methods or in accordance withthe reaction sequences described below. The starting materials used inthe preparation (synthesis) of carrier-linked treprostinil prodrugs ofthe invention or precursors thereof are known or commercially available,or can be prepared by known methods or as described below.

All reactions for the synthesis of the carrier-linked treprostinilprodrugs according to the present invention including precursors are perse well-known to the skilled person and can be carried out understandard conditions according to or analogously to procedures describedin the standard literature of organic chemistry. Depending on thecircumstances of the individual case, in order to avoid side reactionsduring the synthesis of a carrier-linked treprostinil prodrug or aprecursor thereof, it can be necessary or advantageous to temporarilyblock functional groups by introducing protective groups and todeprotect them in a later stage of the synthesis, or introducefunctional groups in the form of precursor groups which in a laterreaction step are converted into the desired functional groups. Suchsynthesis strategies and protective groups and precursor groups whichare suitable in an individual case are known to the skilled person. Ifdesired, the carrier-linked treprostinil prodrugs or precursors thereofcan be purified by customary purification procedures, for example byrecrystallization or chromatography.

In one embodiment, the carrier-linked treprostinil prodrugs according tothe present invention (or a pharmaceutically acceptable salt thereof)may be prepared by a method comprising the steps of converting thecarboxylic acid of the treprostinil to a biologically active moietyreagent D-Y, wherein Y is a leaving group, and subsequently reacting thereagent D-Y with a hydroxyl-group containing reversible prodrug linkerreagent X⁰—OH, thus generating a biologically active moiety-reversibleprodrug linker conjugate T—X⁰ by forming a carboxylic ester linkage.Afterwards, T—X⁰ may be bound to a carrier moiety Z¹ to obtain thecarrier-linked treprostinil prodrug of a biologically active moietycomprising a carboxylic acid group according to the present invention.Alternatively, the carrier moiety Z¹ may already be bound to X⁰—OH.

It is understood that functional groups of treprostinil not involved inthe synthesis of the carrier-linked treprostinil prodrugs of the presentinvention may be protected with suitable protecting groups known to theperson skilled in the art.

Y is a leaving group. Suitable leaving groups are known to a personskilled in the art. Preferably, if attached to D, Y is chloride,bromide, fluoride, nitrophenoxy, imidazolyl, N-hydroxysuccinimidyl,N-hydroxybenzotriazolyl, N-hydroxyazobenzotriazolyl, pentafluorophenoxy,2-thiooxo-thiazolidinyl, or N-hydroxysulfosuccinimidyl.

The carrier-linked treprostinil prodrug of the present invention can beprepared starting from a polymer by convenient methods known in the art.It is clear to a practitioner in the art that several routes exist. Forexample, a moiety T—X⁰ can be reacted with the reactive functionalgroups of the polymer of the carrier moiety POL. Alternatively, areagent comprising a moiety Z¹—X⁰ may be prepared for subsequentreaction with a preferentially activated biologically active acid D-Y.It is understood that D is treprostinil.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention is illustrated hereinafter by drawings and the workingexamples, the drawings and working examples serving merely forillustration but not restricting the invention.

FIG. 1 shows the treprostinil release in buffer and buffered rat plasmaat different time points expressed as % treprostinil release compared tototal treprostinil content (see Example 29).

FIG. 2 shows a prolonged duration of circulation of treprostinilconjugate 25 for more than two weeks in monkeys after subcutaneousinjection (see Example 30).

FIG. 3 shows a single dose iv injection of compound 25 and subsequentplasma analysis for total treprostinil and carrier content (see Example30).

FIG. 4 shows a single dose iv injection of compound 25 and subsequentplasma analysis for free treprostinil (see Example 32).

FIG. 5 shows a single dose subcutaneous injection of compound 25 andsubsequent plasma analysis for free treprostinil (see Example 32).

OPERATIVE EXAMPLES

The subject matter of the present invention is elucidated in more detailbelow, using examples, without any intention that the subject matter ofthe invention should be confined to these exemplary embodiments.

Materials, Methods and Analytics: Product Purification

Normal phase purification was performed on a Biotage “Isolera one”purification system Biotage AB, Sweden. Biotage KP-Sil silicacartridges. Gradients of Heptane/Ethylacetate orDichloromethane/Methanol were used. Products were detected and collectedat 254 and 280 nm.

For preparative RP-HPLC, a Waters 600 controller and a 2487 DualAbsorbance Detector was used equipped with a Waters XBridge™ BEH300 PrepC18 5 μm, 150×10 mm, flow rate 6 ml/min, or Waters XBridge™ BEH300 PrepC18 10 μm, 150×30 mm, flow rate 40 ml/min. Gradients of eluents A (watercontaining 0.05% TFA v/v or 0.01% HCl v/v) and B (acetonitrilecontaining 0.05% TFA v/v or 0.01% HCl v/v) were used.

HPLC fractions containing product were pooled and lyophilized if notstated otherwise.

Automated Flash Chromatography

Automated Flash Chromatography was performed on a Biotage “Isolera one”purification system Biotage AB, Sweden, using Biotage KP-Sil silicacartridges. Products were detected and collected at 254 and 280 nm.

LC/MS Analytics

Analytical RP-HPLC/ESI-MS was performed on waters equipment consistingof a 2695 sample manager, a 2487 Dual Absorbance Detector, and a ZQ 4000ESI instrument equipped with a 5 μm Reprosil Pur 300 Å ODS-3 column(75×1.5 mm) (Dr. Maisch, Ammerbuch, Germany; flow rate: 350 μl/min,typical gradient: 10-90% MeCN in water, 0.05% TFA over 5 min) or on aWaters Acquity UPLC with an Acquity PDA detector coupled to a Thermo LTQOrbitrap Discovery high resolution/high accuracy mass spectrometerequipped with a Waters ACQUITY UPLC BEH300 C18 RP column (2.1×50 mm, 300Å, 1.7 μm, flow: 0.25 mL/min; solvent A: UP-H₂0+0.04% TFA, solvent B:UP-Acetonitrile+0.05% TFA.

RP-UPLC/ESI-MS was performed on Waters/Thermo equipment consisting of aWaters Acquity UPLC with an Acquity PDA detector coupled to a Thermo LTQOrbitrap Discovery high resolution/high accuracy mass spectrometerequipped with a ACQUITY UPLC® BEH300 C18 RP column (Waters Corporation,2.1×50 mm, 300 Å, 1.7 μm, Flow: 0.25 mL/min; solvent A: UP-H₂0+0.04%TFA, solvent B: UP-MeCN+0.05% TFA. Typical gradients for determinationof released treprostinil from TransCon 5 kDa PEG linker treprostinilare: 0.25 mL flow rate, gradient: 30-50% B over 10 min

RP-HPLC Purification:

For preparative RP-HPLC a Waters 600 controller and a 2487 DualAbsorbance Detector was used equipped with the following columns: WatersXBridge™ BEH300 Prep C18 5 μm, 150×10 mm, flow rate 6 ml/min, or WatersXBridge™ BEH300 Prep C18 10 μm, 150×30 mm, flow rate 40 ml/min. Lineargradients of solvent system A (water containing 0.05% TFA v/v or 0.01%HCl v/v) and solvent system B (acetonitrile containing 0.05% TFA v/v or0.01% HCl v/v)

Typical gradients for purification procedures are:

-   -   6 mL/min flow rate, solvent A: H₂0+0.05% TFA, solvent B:        MeCN+0.05% TFA, typical gradient: 1-95% B over 14 min    -   6 mL/min flow rate, solvent A: H₂0+0.05% TFA, solvent B:        MeCN+0.05% TFA, typical gradient: 10-80% B over 14 min    -   40 mL/min flow rate, solvent A: H₂0+0.05% TFA, solvent B:        MeCN+0.05% TFA, typical gradient: 40-95% B over 14 min HPLC        fractions containing product were pooled and lyophilized if not        stated otherwise.

Chemicals and Drug Substances:

Treprostinil acid was purchased from Shanghai Techwell BiopharmaceuticalCo., Ltd., Shanghai, Peoples Republic of China or ChirogateInternational Inc. Yangmei, Taiwan. 6-(S-Tritylmercapto)hexanoic acidwas purchased from Polypeptide, Strasbourg, France.Cis-cyclohexanedicarboxylic anhydride was purchased from Alfa Aesar GmbH& Co KG, Karlsruhe, Germany. 2-Chlorotrityl chloride resin (1%,Novabiochem® DVB) was obtained from Merck Biosciences GmbH, Germany.6-(S-Tritylsulfanyl)-hexaneamine was synthesized according to WO-A2009/133137. PEGs used in this work were acquired from NOF Europe N.V.,Grobbendonk, Belgium. All other chemicals were purchased from SigmaAldrich GmbH, Taufkirchen, Germany. Water and acetonitrile foranalytical RP-HPLC were purchased from Biosolve B.V. and TFA from Thermoscientific.

Example 1 Benzyl Protection of 3-Hydroxybutanoic Acid 1:

3-Hydroxybutanoic acid 1 (434 mg, 4.17 mmol) was dissolved in THF (10mL) and BnBr (700 μL, 5.89 mmol) and Cs₂CO₃ (2.5 g, 7.67 mmol) wereadded. The reaction mixture was refluxed in a sealed tube for 4-6 hours.After cooling down to room temperature the reaction mixture wasfiltrated and the residue was washed several times with EtOAc. Theorganic solvents were removed and the product was purified by automatedflash chromatography on silica in one portion (SNAP 25 g cartridge, flow30 ml/min, solvent A: DCM, solvent B: MeOH; gradient: 0-5% B over 19 CV)to remove starting material and obtain desired benzyl protected3-hydroxybutanoic acid 2 as yellow oil.

Yield: 361 mg (45%)

MS: m/z 217.1=[M+Na]⁺ (MW+Na calculated=217.2).

Example 2

Coupling Reaction of Benzylated 3-Hydroxybutanoic Acid 2 withTreprostinil:

Treprostinil acid (10.5 mg, 0.0268 mmol) was dissolved in DCM (4.5 mL)and DCC (9.4 mg, 0.0455 mmol), HOBT (7.5 mg, 0.0489 mmol) and DMAP (7.5mg, 0.0613 mmol) were added to the solution. Then benzylated3-hydroxybutanoic acid 2 (15 mg, 0.0772 mmol) was dissolved in DCM (0.5mL) and added to the reaction mixture. The mixture was stirred at RTuntil the consumption was complete (analytical RP-HPLC). Volatilesolvents were removed in vacuo and the residue was purified over a smallsilica column (3 ml silica, DCM/MeOH (100:0)—DCM/MeOH (95:5) to obtainthe desired linker treprostinil 3 as yellow oil.

Yield: 8 mg (50%)

MS: m/z 589.3⁼[M+Na]⁺ (MW+Na calculated=589.7)

Example 3 Hydrogenation Reaction of Benzylester 3:

Benzylester 3 (13 mg, 0.0229 mmol) was dissolved in EtOAc (4 Å MS, 2 mL)and 5% palladium on charcoal (5% Pd, 15 mg) was added. Hydrogen wasbubbled through the solution for 30 min. The reaction mixture wasstirred further 12.5 h under hydrogen atmosphere until the consumptionwas complete (analytical RP-HPLC). The mixture was filtered over celiteand washed several times with EtOAc. Organic solvents were removed invacuo and the residue was purified using RP-HPLC (solvent A: H₂O with0.05% TFA, solvent B: MeCN with 0.05% TFA, gradient: 1-95% B over 20min, flow: 6 mL/min). The product containing fractions were pooled andlyophilized to obtain 4 as white solid.

Yield: 1.9 mg (29%).

MS: m/z 499.3=[M+Na]⁺ (MW+Na calculated=499.6).

Example 4

Coupling Reaction of Linear PEG 5 kDa Amine with Linker Treprostinil 4:

Linker treprostinil 4 (1.9 mg, 3.98 μmol) and linear PEG 5 kDa amine (86mg, 17.2 μmol) were dissolved in THF/MeCN (4 Å MS; 1.5 mL: 0.5 mL) andEt₃N(40 μL), a catalytic amount of DMAP and T3P (50% in EtOAc, 50 μL,73.2 μmol) were successively added. The reaction mixture was allowed tostir at rt for 12 h. The reaction mixture was diluted with 20 μL H₂O andvolatile solvents were removed in vacuo. The residue was purified usingRP-HPLC (solvent A: H₂O with 0.05% TFA, solvent B: MeCN with 0.05% TFA,gradient: 10-80% B over 20 min, flow: 6 mL/min). The product containingfractions were pooled and lyophilized to obtain TransCon PEG linkertreprostinil 5 as white solid.

Yield: 12.5 mg (58%).

MS: m/z 1378.6=[M+4H]⁴⁺ (calculated=1378.9) for one representative peakin the polymer distribution.

Example 5 Treprostinil Release Kinetics of TransCon PEG LinkerTreprostinil 5:

TransCon PEG linker treprostinil 5 (0.5-1.5 mg) was incubated in pH 7.4hydrolysis buffer (60 mM sodium phosphate, 3 mM EDTA, 0.05% Tween-20, 1mL) at 37° C. and aliquots were analyzed by UPLC at various time pointsfor released treprostinil.

Half Life Determination of Hydrolysis Kinetics of TransCon PEG LinkerTreprostinil 5:

The percentage of released treprostinil after incubation at pH 7.4 and37° C. for a given time period was determined by integrating thecorresponding peaks (released material versus conjugate) in the RP-UPLCchromatogram. The data as shown in table 1 were subsequently plottedagainst time. By using a first order kinetics fit a half life of 4.20 dfor the treprostinil release from 5 was obtained.

TABLE 1 entry Incubation time [d] released treprostinil [%] 1 0.000 2 20.83 5 3 1.11 18 4 1.81 27 5 2.06 29 6 5.13 59 7 6.10 64 8 8.80 77 911.90 86

Example 6

Synthesis of Intermediates 6a/6b:

6-(S-Tritylsulfanyl)-hexaneamine (for synthesis see WO-A 2009/133137)(507 mg, 1.35 mmol) was dissolved in DCM (4 ml) andcis-1,2-cyclohexanedicarboxylic anhydride (251 mg, 1.63 mmol) was addedto the reaction mixture at RT. DIPEA (0.70 mL, 4.06 mmol) was added andthe mixture was stirred at RT until complete consumption of6-(S-Tritylsulfanyl)-hexaneamine (LC/MS). Volatile solvents were removedin vacuo, the residue was dissolved in H₂O/MeCN (6:1, 18 mL) and theproduct was purified by RP-HPLC (solvent A: H₂O with 0.05% TFA, solventB: MeCN with 0.05% TFA, gradient: 40-95% B over 16 min, flow: 40ml/min). The pooled fractions were neutralized with sat. NaHCO₃ soln.(pH approx. 6) and the organic solvents were removed in vacuo. Theremaining aqueous phase was extracted twice with DCM. Combined organiclayers were dried with MgSO₄ and the solvent was removed in vacuoobtaining 6a/6b as a racemic mixture.

Yield: 580 mg (81%).

MS: m/z 552.23=[M+Na]⁺ (MW+Na calculated=552.62 g/mol).

Example 7

Synthesis of Intermediates 7a/7b:

N-Boc-1,6-hexanediamine (270 mg, 1.25 mmol) was dissolved in DMF (2 ml)and cis-1,2-cyclohexanedicarboxylic anhydride (231 mg, 1.50 mmol) wasadded to the reaction mixture at RT. DIPEA (0.65 mL, 3.76 mmol) wasadded and the mixture was stirred at RT until consumption ofN-Boc-1,6-hexanediamine (LC/MS). The reaction mixture was diluted withH₂O/MeCN (9:1) and the product was purified by RP-HPLC (solvent A: H₂Owith 0.05% TFA, solvent B: MeCN with 0.05% TFA, gradient: 10-80% B over16 min, flow: 40 ml/min). The pooled fractions were neutralized withsat. NaHCO₃ soln. (pH approx. 6) and the organic solvents were removedin vacuo. The remaining aqueous phase was extracted several times withDCM. The organic layers were dried with MgSO₄ and the solvent wasremoved in vacuo obtaining 7a/7b as a racemic mixture.

Yield: 410 mg (88%).

MS: m/z 371.39=[M+H]⁺ (MW+H calculated=371.27 g/mol).

Example 8 Synthesis of Dmob Protected Treprostinil 8:

Treprostinil (61 mg, 0.156 mmol) was dissolved in toluene (dry,molecular sieve, 2.5 ml) and silylation reagent BSA (0.6 mL, 0.245 mmol)was added. The reaction mixture was stirred for 12 h at RT. Volatilesolvents were removed in vacuo and the TMS protected treprostinil wasused without further purification.

TMS protected treprostinil was dissolved in DCM (2.5 mL) and H₂O (60μL). DMAP (76 mg, 0.624 mmol), EDC.HCl (119 mg, 0.624 mmol) andDmob-alcohol (105 mg, 0.624 mmol) dissolved in DCM (1 ml) were added.The reaction mixture was stirred at RT until reaction was complete(LC/MS). The solution was diluted with DCM and quenched by addition of0.1 N HCl solution saturated with NaCl. The aqueous phase was extractedseveral times with DCM. Combined organic layers were dried with MgSO₄and the solvent was removed in vacuo obtaining crude product 8. Crudeproduct was purified using RP-HPLC (solvent A: H₂O with 0.05% TFA,solvent B: MeCN with 0.05% TFA, gradient: 35-85% B over 16 min, flow: 40ml/min). Combined HPLC fractions were adjusted to a pH of approx. 7 byadding sat. NaHCO₃ soln. MeCN was removed in vacuo. The remaining H₂Olayer was extracted several times with DCM and the combined organicphases were dried with MgSO₄, filtered and the solvent was removed invacuo obtaining product 8 as colorless solid.

Yield: 69 mg (82%).

MS: m/z 563.20 g/mol=[M+Na]⁺ (MW+Na calculated=563.67 g/mol).

Example 9 Synthesis of Treprostinil Linker Thiol

Carboxylic acid 6a/6b (147 mg, 0.277 mmol), EDC.HCl (53 mg, 0.277 mmol)and DMAP (34 mg, 0.277 mmol) were dissolved in 0.5 mL DCM. Dmobprotected treprostinil 8 (43 mg, 0.08 mmol) was dissolved in 0.5 mL DCMand added to the reaction mixture. The mixture was stirred at RT untilconsumption of 8 was complete (over night, LC/MS). Volatile solventswere removed in vacuo. The residue was dissolved in HFIP (2 mL), TFA(100 μL) and TES(50 μL) and stirred for 30 min at RT (LC/MS). Volatileswere removed in vacuo. The residue was dissolved in H₂O/MeCN (9/1, 0.05%TFA, 2 mL) and the mixture of four possible isomers was purified byRP-HPLC (solvent A: H₂O with 0.05% TFA, solvent B: MeCN with 0.05% TFA,gradient: 60-85% B over 16 min, flow: 6 mL/min). Product isomers elutedas three separable peaks. Fractions containing the peak with theshortest elution time (compound “9×”) were pooled and used in thePEGylation step without further processing. Structural assignment of 9×to the possible isomers 9a, 9b, 9c or 9d was not performed in thisexperiment. Yield of 9× was determined by using Ellman test.

Yield: 8.1 mg (26%)

MS: m/z 682.21 g/mol=[M+Na]⁺ (MW+Na calculated=682.40 g/mol).

Example 10 Synthesis of Treprostinil Linker Amine

Carboxylic acid 7a/7b (50 mg, 0.134 mmol), EDC HCl (26 mg, 0.134 mmol)and DMAP (16 mg, 0.134 mmol) were dissolved in DCM (0.3 mL). Dmobprotected treprostinil 8 (36 mg, 0.066 mmol) was dissolved in DCM (0.5mL) and added to the reaction mixture. The mixture was stirred at RTuntil the consumption was complete (LC/MS). Volatile solvents wereremoved in vacuo. The residue was dissolved in H₂O/MeCN (9/1, 0.05% TFA,2 mL) and the mono coupling products (treprostinil coupled to one 7a/7bmolecule) were separated from the double coupling products (treprostinilcoupled to two 7a/7b molecules) by RP-HPLC: Thermo Fisher Hypersil GoldPFP column, 150×10 mm, solvent A: H₂O with 0.05% TFA, solvent B: MeCNwith 0.05% TFA, gradient: 35-55% B over 16 min, flow: 6 mL/min. HPLCfractions containing mono coupling products were pooled and lyophilized.Lyophilizate was dissolved in HFIP (0.9 mL), DCM (0.1 mL), TFA (100 μL)and TES(20 μL) and stirred for 10 min at RT. Volatiles were removed invacuo, the residue was dissolved in H₂O/MeCN (9/1, 0.05% TFA, 2 mL) andthe and the mixture of four possible isomers was purified by RP-HPLC(solvent A: H₂O with 0.05% TFA, solvent B: MeCN with 0.05% TFA,gradient: 35-55% B over 16 min, flow: 6 mL/min). Product isomers elutedas three separable peaks. Fractions containing the peak with theshortest elution time (compound “10×”) were pooled and used in thePEGylation step without further processing.

Structural assignment of the 10× to the possible isomers 10a, 10b, 10cor 10d was not performed in this experiment. Yield of 10× was estimatedby HPLC by using a treprostinil calibration curve (280 nm).

Yield: 3.0 mg

MS: m/z 643.28 g/mol=[M+Na]⁺ (MW+Na calculated=643.45 g/mol).

Example 11

PEGylation Reaction of Treprostinil Linker Amine with Linear PEG 5 kDaNHS

To treprostinil linker amine 10× (0.6 mg, 1 μmol in solution, MeCN/H₂O,0.05% TFA, 5 mL) linear PEG 5 kDa NHS (23 mg, 4.6 μmol) was added. Thesolution was neutralized by addition of 0.5 M pH 7.4 buffer (0.5 Mphosphate, 0.6 mL). H₂O (1 mL) was added for obtaining a clear solution,and reaction mixture was incubated at RT for 1 h. Then the reactionmixture was purified by RP-HPLC (solvent A: H₂O with 0.01% HCl, solventB: MeCN with 0.01% HCl, gradient: 10-70% B over 16 min, flow: 6 mL/min)to obtain after lyophilization TransCon linear 5 kDa PEG treprostinil11.

Yield: 3 mg

Example 12

PEGylation Reaction of Treprostinil Linker Thiol with Linear PEG 40 kDaMaleimide

To the treprostinil linker thiol 9× (6.2 mg, 9.42 μmol) solution inMeCN/H₂O (0.05% TFA, 87 mL) linear PEG 40 kDa maleimide (463 mg, 11.3μmol) was added. The solution was neutralized by addition of pH 7.4buffer (0.5 M phosphate, 4.4 mL). After 1 h incubation time anotherportion of linear 40 kDa Mal-PEG (73 mg, 178 μmol) and H₂O (5 mL) wasadded and the reaction solution was incubated for another 1.5 h. Thereaction mixture was purified by RP-HPLC (solvent A: H₂O with 0.01% HCl,solvent B: MeCN with 0.01% HCl, gradient: 30-50% B over 16 min, flow: 40mL/min) to obtain after lyophilization TransCon linear 40 kDa PEGtreprostinil 12.

Yield: 321 mg (82%)

Example 13

PEGylation Reaction of Treprostinil Linker Thiol with 4-Arm PEG 20 kDaMaleimide

To the treprostinil linker thiol 9× (2.54 mg, 3.84 μmol) solution inMeCN/H₂O (0.05% TFA, 5.7 mL) 4-arm PEG 20 kDa maleimide (21 mg, 0.98μmol) was added. The solution was neutralized by addition of pH 7.4buffer (0.5 M phosphate, 3.0 mL). H₂O (3 mL) was added until thereaction mixture became a clear solution again. The reaction mixture wasincubated at RT for 2 h and then purified by RP-HPLC (solvent A: H₂Owith 0.01% HCl, solvent B: MeCN with 0.01% HCl, gradient: 45-85% B over16 min, flow: 40 mL/min) to obtain after lyophilization TransCon 4-armPEG 20 kDa treprostinil 13.

Yield: 14 mg (66%).

Example 14 Treprostinil Release Kinetics of TransCon PEG LinkerTreprostinil Compounds 11 and 12:

Release kinetics were determined according to Example 5. A treprostinilrelease half life time of 4.3 days (±0.7 days) was obtained forcompounds 11 and 12.

Example 15 Treprostinil Release Kinetics of TransCon PEG LinkerTreprostinil Compound 13

TransCon PEG linker treprostinil 13 (2.5 mg) was incubated in pH 7.4hydrolysis buffer (60 mM sodium phosphate, 3 mM EDTA, 0.05% Tween-20, 1mL) at 37° C. and aliquots were analyzed by UPLC at various time pointsfor released treprostinil. The percentage of released treprostinil wasdetermined in relation to the area of treprostinil after totalhydrolysis of an aliquot (50 μl hydrolysis solution and 25 μl 5 N NaOHwere mixed for 20 min. 25 μl AcOH was added and the resulting solutionwas analyzed by LCMS).

By using a first order kinetics fit, a half life of 5 d for treprostinilrelease from 13 was obtained.

Example 16 Synthesis of Building Block 14

Building block 14 was synthesized according to the following scheme:

Mmt-chloride (3 g, 9.71 mmol) was dissolved in DCM (20 mL) and addeddropwise to a solution of ethylenediamine (6.5 mL, 97.1 mmol) in DCM (20mL). After two hours the solution was poured into diethyl ether (300 mL)and washed three times with 30/1 (v/v) brine/0.1 M NaOH solution (50 mleach) and once with brine (50 mL). The organic phase was dried overNa₂SO₄ and volatiles were removed under reduced pressure. Mmt-protectedamine (3.18 g, 9.56 mmol) was used in the next step without furtherpurification.

The Mmt-protected amine (3.18 g, 9.56 mmol) was dissolved in anhydrousDCM (30 mL). 6-(S-Tritylmercapto)hexanoic acid (4.48 g, 11.47 mmol),PyBOP (5.96 g, 11.47 mmol) and DIPEA (5.0 mL, 28.68 mmol) were added andthe mixture was agitated for 30 min at RT. The solution was diluted withdiethyl ether (250 mL) and washed three times with 30/1 (v/v) brine/0.1M NaOH solution (50 mL each) and once with brine (50 mL). The organicphase was dried over Na₂SO₄ and volatiles were removed under reducedpressure. Amide was purified by flash chromatography eluting withheptane/ethyl acetate containing 0.02% (v/v) diethylmethylamine.

Yield: 5.69 g (8.07 mmol).

MS: m/z 705.4=[M+H]⁺ (MW=705.0).

Amide (3.19 g, 4.53 mmol) was dissolved in anhydrous THF (50 mL) andBH₃.THF (1 M solution, 8.5 mL, 8.5 mmol) was added. Solution was stirredfor 16 h at RT. Further BH₃.THF (1 M solution, 14 mL, 14 mmol) was addedand stirred for further 16 h at RT. The reaction was quenched byaddition of methanol (8.5 mL). N,N-dimethyl-ethylenediamine (3 mL, 27.2mmol) was added, the solution was heated to reflux and stirred for 3 h.Reaction mixture was allowed to cool down to RT and was then dilutedwith ethyl acetate (300 mL), washed with saturated, aqueous Na₂CO₃solution (2×100 mL) and saturated, aqueous NaHCO₃ solution (2×100 mL).The organic phase was dried over Na₂SO₄ and volatiles were removed underreduced pressure to obtain crude amine intermediate (3.22 g).

The amine intermediate (3.22 g) was dissolved in DCM (5 mL). Boc₂O (2.97g, 13.69 mmol) dissolved in DCM (5 mL) and DIPEA (3.95 mL, 22.65 mmol)were added and the mixture was agitated at RT for 30 min. Boc- andMmt-protected intermediate was purified by flash chromatography.

Yield: 3.00 g (3.79 mmol).

MS: m/z 791.4=[M+H]⁺, 519.3=[M−Mmt+H]⁺ (MW calculated=791.1).

0.4 M aqueous HCl (48 mL) was added to a solution of the Boc- andMmt-protected intermediate in acetonitrile (45 mL). The mixture wasdiluted with acetonitrile (10 mL) and stirred for 1 h at RT.Subsequently, the pH value of the reaction mixture was adjusted to 5.5by addition of an aqueous 5 M NaOH solution. Acetonitrile was removedunder reduced pressure and the aqueous solution was extracted with DCM(4×100 mL). The combined organic phases were dried over Na₂SO₄ andvolatiles were removed under reduced pressure. Crude amine 14 was usedwithout further purification.

Yield: 2.52 g (3.19 mmol). A MW of 791.1 g/mol of crude amine 14 wasassumed

MS: m/z 519.3=[M+H]⁺ (MW calculated=519.8 g/mol).

Example 17 Synthesis of Linker Building Blocks 15a, 15b, and 15c

Linker building block 15a was synthesized according to the followingscheme:

Amine 14 (503 mg, 0.635 mmol, assuming a MW of 791.1 g/mol of crude 1)was dissolved in 4 mL DMF (anhydrous, mol. sieve). Fmoc-N-Me-Ala-OH (310mg, 0.953 mmol), COMU (408 mg, 0.953 mmol) and DIPEA (332 μl, 1.906mmol) were added and the reaction was allowed to stir for 3 h at RT. 150μl piperidine and 150 μl DBU were added to the mixture and stirring wascontinued for further 60 min. 400 μl acetic acid were added and productwas purified by HPLC. HPLC fractions containing product 15a wereneutralized with a saturated NaHCO₃ solution and extracted twice withDCM. Combined organic phases were dried over Na₂SO₄ and volatiles wereremoved under reduced pressure.

Yield: 203 mg (0.336 mmol).

MS: m/z 604.1=[M+H]⁺ (MW calculated=603.9 g/mol).

Linker Building Block 15b

Linker building block 15b was synthesized as described for 15a exceptthat Fmoc-Aib-OH was used instead of Fmoc-N-Me-Ala-OH.

Yield: 95 mg (0.161 mmol).

MS: m/z 604.2=[M+H]⁺ (MW calculated=603.9 g/mol).

Linker Building Block 15c

Linker building block 15c was synthesized as described for 15a exceptthat Fmoc-N-Me-Aib-OH was used instead of Fmoc-N-Me-Ala-OH.

Yield: 149 mg (0.241 mmol).

MS: m/z 619.0=[M+H]⁺ (MW calculated=617.9 g/mol).

Example 18 Synthesis of Treprostinil-Linker Thiols 16a, 16b, 16c, 16d,16e and 16f

Treprostinil-linker thiols 16a/16b were synthesized according to thefollowing scheme:

A 10 mL single use syringe reactor equipped with a PE frit was loadedwith 2-chlorotrityl chloride (TCP) resin (153 mg, loading 1.22 mmol/g,0.186 mmol). A solution of treprostinil (54 mg, 0.138 mmol) and DIPEA(60 μl, 0.346 mmol) in DCM (anhydrous, mol. sieve) was drawn into thereactor. Reactor was agitated for 2 h at RT. 200 μl methanol were addedand reactor was agitated for further 10 min. Solution was dispelled andresin was washed with DCM (5×), DMF (5×) and DCM (10×). Resin was driedunder vacuum (1 mbar). Based on weight, a treprostinil loading of 0.72mmol/g TCP resin was obtained.

900 μl THF (anhydrous, mol. sieve) and 300 μl of a 1 M LiOEt solution inTHF (300 μmol) were drawn to 30 mg treprostinil loaded TCP resin (21.6μmol) in a single use 2 mL syringe reactor equipped with a PE frit.Reactor was agitated for 40 min at RT. Solution was dispelled and resinwas washed with THF (2×). A solution of bis(pentafluorophenyl)carbonate(100 mg, 254 μmol) in 1 mL THF was drawn into the syringe which wasagitated for 90 min at RT. Solution was dispelled and resin was washedwith THF (5×) and DMF (5×). A solution of linker building block 15a (50mg, 83 μmol), DIPEA (50 μl, 287 μmol) and DMAP (1 mg, 8 μmol) in 300 μlDMF (anhydrous, mol. sieve) was drawn into the syringe. Syringe wasagitated for 3 h at RT. Solution was dispelled and resin was washed withDMF (10×) and DCM (10×). Product was cleaved from resin by incubationwith 500 μl of cleavage cocktail HFIP/DCM/TES 90/10/2 v/v/v for 10 min(3×). Resin was washed with 500 μl DCM (2×). TFA (250 μL) was added tothe combined cleavage and washing solutions and the mixture wasincubated at RT for 10 min. Volatiles were removed under reducedpressure. Residue was subjected to HPLC purification which gave thiols16a/16b as a mixture of the two regioisomers. HPLC eluate was used inthe next step without further processing.

MS: m/z 678.1=[M+H]⁺ (MW calculated=678.0 g/mol).

Treprostinil Linker Thiols 16c/16d

Treprostinil linker thiols 16c/16d were synthesized as described for16a/16b except that linker building block 15b was used instead of 15a.Thiols 16c/16d were obtained as a mixture of isomers. HPLC eluate wasused in the next step without further processing.

MS: m/z 678.1=[M+H]⁺ (MW calculated=678.0 g/mol).

Treprostinil Linker Thiols 16e and 16f

Treprostinil linker thiols 16e and 16f were synthesized as described for16a/16b except that linker building block 15c was used instead of 15a.Two isomers assigned to structures 16e and 16f were separated by HPLC.HPLC eluates were used in the next step without further processing.

15e MS: m/z 693.0=[M+H]⁺ (MW calculated=692.0 g/mol).

15f MS: m/z 693.0=[M+H]⁺ (MW calculated=692.0 g/mol).

Example 19

Synthesis of Linker Building Blocks 17a□ and 17b

Linker building blocks 17a and 17b were synthesized according to thefollowing scheme:

L-Fmoc-Dpr(Boc)-OH (100 mg, 0.234 mmol) was dissolved in 0.5 mL DMF(anhydrous, mol. sieve). 6-(S-Tritylsulfanyl)-hexaneamine (71 mg, 0.189mmol), COMU (97 mg, 0.227 mmol) and DIPEA (66 μl, 0.378 mmol) were addedand mixture was stirred for 1 h at RT. Piperidine (50 μl, 0.505 mmol)and DBU (40 μl, 0.336 mmol) were added and stirring was continued for 10h. ds-Cyclohexanedicarboxylic anhydride (600 mg, 3.89 mmol) was addedand stirring was continued for 1 h. Solution was quenched withwater/acetonitrile and acidified with acetic acid Building blocks werepurified by RP-HPLC. Structures assignment of the earlier elutingdiastereomer 17a and the later eluting diastereomer 17b was donearbitrarily and could also be reverse.

Yield: 17a 30 mg (0.042 mmol), 17b 42 mg (0.059 mmol)

MS: m/z 716.2=[M+H]⁺ (MW calculated=716.0 g/mol).

Example 20

Synthesis of Treprostinil Linker Thiols 18a/18b

Linker building block 17a (11 mg, 12 μmol), EDC HCl (7.4 mg, 38.5 μmol)and DMAP (4.7 mg, 38.5 μmol) were dissolved in 300 μl DCM (anhydrous,mol. sieve). Solution was drawn to 15 mg treprostinil loaded TCP resin(10.8 μmol, 0.72 mmol/g see Example 3) in a single use 2 mL syringereactor equipped with a frit. Reactor was agitated for 15 h at RT.Solution was dispelled and resin was washed with DCM (10×). Product wascleaved by incubating resin with 500 μl HFIP/DCM 30/70 v/v for 10 min(3×). Resin was washed with 500 μl DCM (2×). To the combined cleavageand washing solutions were added 250 μl TFA and the mixture wasincubated at RT for 10 min. Volatiles were removed under reducedpressure. Residue was subjected to RP-HPLC purification which gavethiols 18a/18b as a mixture of the two regioisomers. HPLC eluate wasused in the next step without further processing.

Yield: 18a/18b 1.5 mg (2 μmol) as determined by thiol quantification byEllman Test.

MS: m/z 746.2=[M+H]⁺ (MW calculated=746.0 g/mol).

Example 21

Synthesis of PEG-Linker-Drug Conjugates 19a/b, 19c/6d, 19e, 19f and19g/19h

PEG-linker-drug conjugates were prepared according to the followingscheme:

To HPLC eluates of treprostinil linker thiols 16a/16b, 16c/16d, 16e, 16fand 18a/18b was given an excess of linear PEG 5 kDa maleimide. Mixtureswere neutralized by addition of pH 7.4 buffer (0.5 M phosphate) andincubated at RT. After complete consumption of thiol (approx. 1 h)mixtures were acidified with acetic acid and separated from excessPEG-maleimide by RP-HPLC. HPLC eluates were lyophilized to yieldPEG-linker-drug conjugates 19a/b, 19c/19d, 19e, 19f and 19g/19hrespectively.

Example 22

Determination of Drug Release Half Life Time from PEG Conjugates 19a/b,19c/19d, 19e, 19f and 19g/19h:

PEG-linker-drug conjugates 19a/b, 19c/19d, 19e, 19f and 19g/19h weredissolved in pH 7.4 buffer (60 mM sodium phosphate, 3 mM EDTA, 0.05%Tween-20, 1 mL) and incubated at 37° C. At various time points aliquotswere analyzed by UPLC to determine the amount of released treprostinilwhich was plotted against time. Drug release was found to follow firstorder kinetics. Curve fitting software was used to determine half lifetime of drug release from the respective conjugates (Table 1)

TABLE 2 entry PEG-linker-drug conjugate drug release half life time 119a/b  31 d 2 19c/19d 17 d 3 19e 24 d 4 19f 37 d 5 19g/19h 35 min

Example 23 Synthesis of Intermediate 20:

The amino group of 6-(S-Tritylsulfanyl)-hexaneamine was Tmob(2,4,6-Trimethoxybenzyl) protected by dropwise addition of a solution of2,4,6-trimethoxybenzaldehyde (4.22 g, 21.51 mmol) in 88 mL methanol/DCM1/1 (v/v) to 6-(S-tritylsulfanyl)-hexaneamine (6.74 g, 17.95 mmol) andsodium cyanoborohydride (1.58 g, 25.14 mmol) in 44 mL methanol. Themixture was stirred for 1.5 h at RT and quenched with 95 mL of 0.4 Naqueous HCl solution. After further stirring at RT for 30 min mixturewas extracted with ethyl acetate (4×). Combined organic layers werewashed with sat. aqueous NaHCO₃ solution (2×) and brine. The organiclayer was dried over Na₂SO₄ and the solvent was removed under reducedpressure. Tmob protected amine 20 was purified by flash chromatographyeluting with DCM/methanol containing 0.1% (v/v) triethylamine.

Yield: 5.88 g (55%).

MS: m/z 556.3⁼[M+H]⁺ (MW calculated=555.79 g/mol).

Example 24 Synthesis of Intermediate 21:

(1R,2S)-Cyclohexanedicarboxylic acid 1-methyl ester, CAS no. 88335-92-6(for synthesis see R. Manzano et al. J. Org. Chem. 2010, 75(15),5417-5420) (506 mg, 2.72 mmol) was dissolved in toluene (11 ml,anhydrous). Thionyl chloride (1.09 mL, 15.0 mmol) was added and mixturewas heated for 1 h at 60° C. in a pressure tube. Volatiles were removedin vacuo. A solution of Tmob protected amine 20 (1.66 g, 2.99 mmol) andDIPEA (1.12 mL, 6.43 mmol) in DCM (30 mL, anhydrous) was added andmixture was stirred for 2 h at RT. Ethyl acetate was added and theorganic layer was washed with 0.1 N aqueous HCl (2×). The organic layerwas dried over Na₂SO₄ and the solvent was removed under reducedpressure. Methyl ester 21 was purified by flash chromatography elutingwith ethyl acetate/heptane.

Yield: 1.55 g (79%).

MS: m/z 746.1=[M+Na]⁺ (MW calculated=723.98 g/mol).

Example 25 Synthesis of Intermediate 22:

Methyl ester 21 (3.12 g, 4.31 mmol) was dissolved in isopropanol (10ml). 35 mL of a 1 M aqueous LiOH solution were added and the mixture wasstirred for 5 d at RT. Ethyl acetate was added and the organic layer waswashed with 0.05 N aqueous HCl (2×) and brine. The organic layer wasdried over Na₂SO₄ and the solvent was removed under reduced pressure. 22was purified by flash chromatography eluting with ethyl acetate/heptanecontaining 0.1% formic acid (v/v).

Yield: 2.41 g (79%).

MS: m/z 710.1=[M+H]⁺ (MW calculated=709.95 g/mol).

Example 26 Synthesis of Intermediate 6a:

Compound 22 (1.23 g, 1.74 mmol) was dissolved in DCM (18 ml). TFA (2 mL)and TES (600 μl) were added and the mixture was stirred for 40 min atRT. Volatiles were removed in vacuo. The residue was dissolved in DCM(20 mL) and tritylchloride (728 mg, 2.61 mmol) was added. The mixturewas stirred for 2 h at RT. DCM was removed under reduced pressure.Carboxylic acid 6a was purified by flash chromatography using ethylacetate/heptane containing 0.1% formic acid (v/v) as eluent, followed byRP-HPLC purification.

Yield: 615 mg (67%).

MS: m/z 552.2=[M+Na]⁺ (MW calculated=⁵²⁹0.7⁵ g/mol).

Example 27 Synthesis of Treprostinil Linker Thiol 24a

Dmob protected treprostinil 8 (100 mg, 0.185 mmol), carboxylic acid 6a(195 mg, 0.368 mmol), EDC.HCl (72 mg, 0.376 mmol) and DMAP (43 mg, 0.352mmol) were dissolved in DCM (1.8 mL, anhydrous, mol. sieve). The mixturewas stirred at RT for 1 d. Ethyl acetate was added and the organic layerwas washed with 0.1 N aqueous HCl (3×) and brine. The organic layer wasdried over Na₂SO₄ and the solvent was removed under reduced pressure.The residue was dissolved in HFIP (5 mL), TFA (250 μL) and TES(250 μL)and stirred for 30 min at RT. The precipitate was filtered off and thefiltrate was evaporated in vacuo.

UPLC analysis revealed a 4/1 ratio of regioisomers 24a and 24b (column:Kinetex 100×2.1 mm, 1.7 μm XB-C18 silica, pore size 100 Å, PhenomonexLtd, Aschaffenburg, Germany; flow rate 0.25 mL/min; solvent A:water+0.05% TFA (v/v), solvent B: acetonitrile+0.04% TFA; gradient:30-58% B (10 min), 58% B isocratic (10 min), 58-80% B (5 min), 80-99% (5min), wavelength 280 nm). 24a turned out to be identical with compound9×.

The residue was taken up in acetonitrile/water and 24a was purified byRP-HPLC (solvent A: H₂O+0.01% HCl, solvent B: MeCN+0.01% HCl, gradient:60-85% B over 16 min). Isomer 24a eluted first, followed by isomer 24b.Fractions containing pure 24a were combined and lyophilized. Mixedfractions containing 24a and 24b were subjected to repurification.

Yield 24a: 29.5 mg (24%)

MS: m/z 660.3=[M+H]⁺ (MW calculated=659.9 g/mol).

¹H-NMR (CDCl₃, δ [ppm]): 7.07 (t, 1H), 6.80 (d, 1H), 6.71 (d, 1H), 5.86(bs, 1H), 4.78-4.63 (m, 3H), 3.53 (bs, 1H), 3.14-3.03 (m, 1H), 3.03-2.83(m, 2H), 2.82-2.66 (m, 2H), 2.66-2.58 (m, 1H), 2.58-2.46 (m, 4H),2.46-2.31 (m, 1H), 2.31-2.13 (m, 1H), 2.13-1.92 (m, 2H), 1.92-1.81 (m,1H), 1.75-1.51 (m, 7H), 1.51-1.21 (m, 21H), 1.21-1.08 (m, 1H), 0.90 (t,3H).

¹³C-NMR (126 MHz, CDCl₃, δ [ppm]): 174.7, 174.2, 171.7, 155.2, 140.6,127.5, 126.3, 121.8, 109.71, 78.8, 72.4, 65.8, 48.3, 43.9, 42.5, 40.4,39.7, 37.4, 37.3, 35.4, 34.00, 33.0, 32.9, 32.1, 29.3, 28.4, 28.1, 27.0,26.4, 25.5, 24.6, 24.2, 23.2, 22.8, 14.2.

Example 28

PEGylation Reaction of Treprostinil Linker Thiol 24a with 4-Arm PEG 20kDa Maleimide

A solution of treprostinil linker thiol 24a (7.5 mg, 11.3 μmol) in 2 mLof acetonitrile/water 9/1 (v/v) was mixed with a solution of 4-arm PEG20 kDa maleimide (53.5 mg, 2.54 μmol) in 2 mL of acetonitrile/water 1/1(v/v). The pH was adjusted to 7.0 by addition of pH 7.4 buffer (50 mMphosphate, 0.8 mL). The reaction mixture was stirred at RT for 1.5 h andthen purified by RP-HPLC (solvent A: H₂O with 0.01% HCl, solvent B: MeCNwith 0.01% HCl, gradient: 45-85% B over 16 min). Product containingfractions were pooled and acetonitrile was removed under reducedpressure. The solution was neutralized by addition of pH 7.4 buffer(phosphate, 0.5 M). The solution was concentrated and the buffer wasexchanged with 10 mM pH 7.0 phosphate containing 46 g/l mannitol byultrafiltration (Vivaspin centrifugal concentrator, PES membrane with 10kDa cut off) to obtain 8.5 mL of the final solution of 25. UPLC and SECanalysis revealed a uniform material. The concentration was determinedby quantification of treprostinil content after basic hydrolysis: Laliquots were treated with 35 μL 0.5 M NaOH. After 30 min incubation atRT 35 μL acetic acid was added. The trerostinil content was determinedby UPLC by using a treprostinil calibration curve. A total treprostinilcontent of 2.0 mg was found, corresponding to 30 mg 25. Yield: 50% basedon PEG starting material.

Example 29 Treprostinil Release Kinetics of TransCon PEG LinkerTreprostinil Compound 25

Treprostinil release kinetics from 25 was determined as described inexample 15 and compared with the results obtained from compound 13. Nodifference in half life time (5 d) was observed.

Example 30 Treprostinil Release Kinetics of TransCon PEG LinkerTreprostinil Compound 13 in Rat Plasma

150 μl of a pH 7.5 HEPES buffer (1 M HEPES, 3 mM EDTA) were mixed with1.2 mL rat plasma (WISTAR rat Li heparin plasma, Innovative Research,Novi, Mich., USA). 150 μl of a of TransCon PEG linker treprostinil 13solution (0.15 mg 13 in 1.5 mL 10 mM phosphate 46 g/l mannitol buffer pH7.0) were added. A pH of 7.4 of the mixture was confirmed by means of apH electrode. Mixture was incubated at 37° C. At given time points 100μl aliquots were withdrawn. 100 μl aliquots were analyzed for releasedand total treprostinil content.

For analysis of released treprostinil, 100 μl aliquots were spiked with20 μl internal standard (2.8 μg/mL tolbutamide in methanol/water 1/1(v/v)) and transferred to a Ostro 96 well plate (Waters GmbH, Eschborn,Germany). Plasma proteins were precipitated by addition of three volumesof pre-cooled (0-5° C.) acetonitrile containing 1% formic acid. Positivepressure was applied (4 bar, Waters Positive Pressure-96 Processor) andeluate was lyophilized. Lyophilizate was dissolved in 40 μl of 10 mMammonium formiate pH 4.0/acetonitrile 7/3 (v/v). Solution wascentrifuged and supernatant was assayed for released treprostinil byUPLC-MS/MS.

For analysis of total treprostinil content (sum of released and carrierbound treprostinil), 100 l aliquots were spiked with 20 μl internalstandard (2.8 μg/mL tolbutamide in methanol/water 1/1 (v/v)) and 50 μlof 0.5 M LiOH were added. Mixture was incubated in a shaker for 2 h atroom temperature. After addition 25 μL 1 M HCl the mixture wastransferred to a Ostro 96 well plate (Waters GmbH, Eschborn, Germany).Plasma proteins were precipitated by addition of three volumes ofpre-cooled (0-5° C.) acetonitrile containing 1% formic acid. Positivepressure was applied (4 bar, Waters Positive Pressure-96 Processor) andeluate was lyophilized. Lyophilizate was dissolved in 100 μl of 10 mMammonium formiate pH 4.0/acetonitrile 7/3 (v/v). Solution wascentrifuged and supernatant was assayed for total treprostinil contentby UPLC-MS/MS.

UPLC-MS/MS method for determination of treprostinil content:

The quantification of plasma treprostinil concentrations were carriedout using a Waters Acquity UPLC coupled to a Thermo LTQ OrbitrapDiscovery mass spectrometer via an ESI probe and with Waters BEH C18(50×2.1 mm I.D., 1.7 μm particle size) as analytical column (mobilephase A: 10 mM ammonium formate pH 4.6, mobile phase B: methanol, T=22°C.). The gradient system comprised a linear gradient from 0.1% B to 95%B in 4 min, an isocratic washing phase with 95% B (0.5 min), and areconditioning phase (2.4 min) with a flow rate of 0.25 mL/min.Detection of the ions was performed in the selected reaction monitoring(SRM, negative ionization) mode, monitoring the transition pairs at them/z 389.2 precursor ions to the m/z 331.2 product ions for treprostiniland m/z 269.1 precursor ions to the m/z 170.0 product ions for theinternal standard (IS) tolbutamide.

The calibration curve was acquired by plotting the extracted peak arearatio area_(treprostinil)/area_(tolbutamide) against the nominaltrepostinil concentrations of calibration standards. The results werefitted to linear regression using standard software.

The extracted peak area ratio area_(treprostinil)/area_(tolbutamide) ofthe quantification experiments at different time points were used tocalculate the treprostinil content according to the calibration curve.

Treprostinil release at time points was expressed as % treprostinilrelease compared to total treprostinil content (see FIG. 1). By using afirst order kinetics fit a half life of 4.5 d for the release kineticsof treprostinil from 25 in buffered rat plasma at 37° C. was obtained,which is in good agreement to release kinetics in pH 7.4 buffer at 37°C. (Example 15).

Example 31 PK of PEG Treprostinil Conjugate 25 in Monkeys

25 (3 mg/mL in 10 mM pH 7.0 phosphate, 46 g/L mannitol) was given at adose level of 0.5 mg/kg as a single dose by sc and iv injection in threemale cynomolgus monkeys each. Blood samples were collected at given timepoints over two weeks. The plasma was assayed for PEG content and totaltreprostinil content (sum of released and carrier bound treprostinil).Due to the fast elimination of free treprostinil compared to carrierbound treprostinil, treprostinil plasma levels reflect the presence oftreprostinil conjugate rather than free treprostinil levels.

For the analysis of total treprostinil content, 100 μL plasma samplesand treprostinil standards in cynomolgus monkey plasma were spiked with20 μL internal standard (2.8 μg/mL tolbutamide in methanol/water 1/1(v/v)) and 50 μl of 0.5 M LiOH were added. The mixture was incubated ina shaker at RT for 2.5 h. After addition of 25 μL 1 M HCl the mixturewas transferred to an Ostro 96 well plate (Waters GmbH, Eschborn,Germany). Plasma proteins were precipitated by addition of three volumesof pre-cooled (0-5° C.) acetonitrile containing

1% formic acid. Positive pressure was applied (4 bar, Waters PositivePressure-96 Processor) and the eluate was lyophilized. The lyophilizatewas dissolved in 100 μl of 10 mM ammonium formiate pH 4.0/acetonitrile7/3 (v/v). The solution was centrifuged and the supernatant was assayedfor total treprostinil content by UPLC-MS/MS.

UPLC-MS/MS method for determination of treprostinil content:

The quantification of plasma treprostinil concentrations were carriedout using a Waters Acquity UPLC coupled to a Thermo LTQ OrbitrapDiscovery mass spectrometer via an ESI probe and with a Waters BEH C18(50×2.1 mm I.D., 1.7 μm particle size) as analytical column (mobilephase A: 10 mM ammonium formate pH 4.6, mobile phase B: methanol, T=22°C.). The gradient system comprised a linear gradient from 0.1% B to 95%B in 4 min, an isocratic washing phase with 95% B (0.5 min), and areconditioning phase (2.4 min) with a flow rate of 0.25 mL/min.Detection of the ions was performed in the selected reaction monitoring(SRM, negative ionization) mode, monitoring the transition pairs at them/z 389.2 precursor ions to the m/z 331.2 product ions for treprostiniland m/z 269.1 precursor ions to the m/z 170.0 product ions for theinternal standard (IS) tolbutamide.

The calibration curve was acquired by plotting the extracted peak arearatio area_(treprostinil)/area_(tolbutamide) against the nominaltrepostinil concentrations of calibration standards which were preparedin cynomolgus monkey plasma. The results were fitted to a linearregression using standard software.

The extracted peak area ratio area_(treprostinil)/area_(tolbutamide) ofthe quantification experiments at different time points were used tocalculate the treprostinil content according to the calibration curve.

For analysis of total PEG content, plasma samples underwent basicpreincubation in order to generate a uniform PEG material from 25. Thiswas based on the fact that after injection of 25 different treprostinilcarrier species are generated due to the sequential release of 4treprostinils from carrier molecule over time.

50 μl plasma samples and 25 PEG treprostinil conjugate standards incynomolgus monkey plasma were diluted with 50 μL of 200 mM HEPESsolution (pH 7.5) and 50 μl of 0.5 M LiOH were added. Mixture wasincubated in a shaker for 2 h at room temperature. After addition of 50μL 1 M HCl the mixture was assayed using the high sensitivity PEG ELISAkit P-0003 from Life Diagnostics Inc. West Chester, Pa., USA, accordingto the manufacturer's instructions.

The calibration curve was acquired by plotting the absorption values at450 nm against the nominal PEG concentrations of calibration standards.The results were fitted to a sigmoidal curve using standard software.

The absorption values at 450 nm of the quantification experiments atdifferent time points were used to calculate the PEG content accordingto the calibration curve.

Result: Total treprostinil content analysis after a single dose scinjection of 25 reveals a prolonged duration of circulation oftreprostinil conjugate for more than two weeks in monkeys. (FIG. 2)

Single dose iv injection of 25 and plasma analysis for totaltreprostinil content revealed similar duration of circulation (FIG. 3).An apparent first order total treprostinil elimination half life time of2.9 days (rate constant k_(apparent): 0.239 d⁻¹) was obtained by usingstandard software.

In contrast plasma analysis for PEG carrier content revealed a muchslower elimination (FIG. 3). By fitting as a first order kinetics usingstandard software an PEG carrier elimination half life time of 6.6 d(rate constant k_(PEGelim): 0.105 d⁻¹) was obtained.

Equal elimination rate constant of different treprostinil carrierspecies e.g. generated by sequential linker hydrolysis/release of one tofour treprostinils from PEG carrier are assumed.

The apparent faster elimination half life time of total treprostinilcompared to PEG carrier is based on the combination of elimination ofthe PEG carrier and treprostinil release by linker hydrolysis. From thedetermined rate constant values of k_(apparent) (0.239 d⁻¹) andk_(PEGelim) (0.105 d⁻¹) a first order treprostinil release by linkercleavage rate constant k_(linker) can be calculated:

exp(−k _(apparent) t)=exp(−k _(PEGelim) t)*exp(−k _(linker) t)=exp(−t[k_(PEGelim) +k _(linker)])

After logmarithizing and rearrangement k_(linker) can be calculatedaccording to:

k _(linker) =k _(apparent) −k _(PEGelim) ; k _(linker)=0.239 d⁻¹−0.105d⁻¹=0.134 d⁻¹

By help of the equation t_(half life time)=ln(2)/k the half life time oftreprostinil release by linker hydrolysis was calculated as 5.2 d, whichis in good agreement with the 5 d linker treprostinil release half lifetime determined in vitro.

Example 32 PK of PEG Treprostinil Conjugate 25 and Free Treprostinil inRats

25 (3 mg/mL buffer (10 mM pH 7.0 phosphate, 46 g/L mannitol)) wasinjected at a dose level of 5.5 mg/kg as a single dose in male Wistarrats each. Three animals received sc injection and three animalsreceived iv injection. Blood samples were collected at given time pointsover two weeks. Blood samples (250 μL) were given directly intocollection tubes containing 50 μL acidic citrate buffer (0.5 M sodiumcitrate, pH 4.0). The plasma was assayed for free treprostinil contentand total treprostinil content (sum of free and carrier boundtreprostinil).

For the analysis of free treprostinil, 50 μL plasma were thawed on iceand mixed with 5 μl acidic citrate buffer and 10 μL internal standard(0.28 μg/mL tolbutamide in methanol/water 1/1 (v/v)). Samples weretransferred to Ostro 96 well plates (Waters GmbH, Eschborn, Germany),and plasma proteins were precipitated by rapid addition of 400 μLpre-cooled (0-5° C.) acetonitrile containing 1% formic acid. Positivepressure was applied (4 bar, Waters Positive Pressure-96 Processor) andthe eluates were collected. Subsequently, the well plates were rinsedtwo times with 100 μL ice-cold acetonitrile containing 1 vol. % formicacid. The eluates were transferred into 2 mL vials, placed into anEppendorf Thermomixer (at 10° C.) and eluates were concentrated under asoft stream of nitrogen over 45 min to a final volume of 60-80 μL. 30 μlsolvent mixture (10 mM aqueous ammonium formiate adjusted to pH 4.0 withformic acid/acetonitrile 7/3 (v/v)) were added to each vial and thesolutions were analyzed by UHPLC-MS/MS.

For preparation of calibration standards, blank plasma samples werespiked with treprostinil and treated likewise.

UHPLC-MS/MS method for determination of free treprostinil content:

The quantification of plasma treprostinil concentrations were carriedout using an Agilent 1290 UHPLC coupled to an Agilent Triplequad 6460system (MassHunter Xcalibur software) in the ES-mode. As analyticalcolumn a Waters BEH C18 was used (50×2.1 mm I.D., 1.7 μm particle size.Mobile phase A: 10 mM ammonium formate pH 5.7, mobile phase B: methanol.The gradient system comprised a linear gradient from 35% B to 99% B in 8min, an isocratic washing phase with 99% B (0.9 min), and areconditioning phase (3 min) with a flow rate of 0.200 mL/min (T=40°C.).

Detection of the ions was performed in the SRM mode, monitoring thetransition pairs at the m/z 389.1 precursor ions to the m/z 331.1product ions for treprostinil and m/z 269.0 precursor ions to the m/z169.9 product ions for the internal standard (IS) tolbutamide. Thecalibration curve was acquired by plotting the extracted peak area ratioarea treprostinil/area tolbutamide against the nominal trepostinilconcentrations of calibration standards. The results were fitted to alinear regression using standard software.

The extracted peak area ratio area treprostinil/area tolbutamide of thequantification experiments at different time points were used tocalculate the treprostinil content according to the calibration curve.

Total treprostinil plasma content was determined as given in Example 31.

Result: Free and total treprostinil content analysis after a single dosesc injection of 25 reveals a prolonged circulation of treprostinilconjugate and a burstless release of free treprostinil for more thanfour days in rats after iv (FIG. 4) or sc injection (FIG. 5).

Example 33

Isolation of Intermediate 6a by Enantioseparation of Racemic Mixture6a/6b

Racemic mixture 6a/6b (107 g) was separated on a Chiralpak IA column(250×76 mm, 20 μm, flow rate 270 mL/min) using acetonitrile/acetic acid1000/1 (v/v) as eluent. Combined eluates of second eluting enantiomer(6a) were mixed with 5 vol % water and evaporated under reducedpressure. The residue was taken up in DCM (500 mL) and extracted with0.1 M HCl (500 mL, 2×) and brine (500 mL). The organic phase was driedover Na₂SO₄ and the solvent was removed under reduced pressure.

Yield: 28.9 g (27%).

MS: m/z 552.2=[M+Na]⁺ (MW calculated=⁵²⁹0.7⁵ g/mol).

Enantiomeric ratio of 6a/6b as determined by Chiralpak IC column(4.5×250 mm, 5 μm, eluent acetonitrile/acetic acid 1000/1 (v/v), flowrate 1 mL/min, 230 nm): 97.5/2.5

Example 34 Improved Synthesis of Treprostinil Linker Thiol 24a

Dmob protected treprostinil 8 (200 mg, 0.370 mmol), carboxylic acid 23(294 mg, 0.555 mmol), EDC.HCl (248 mg, 1.295 mmol) and DMAP (158 mg,1.295 mmol) were dissolved in DCM (2.9 mL, anhydrous, mol. sieve). Themixture was stirred at RT for 1 d. Ethyl acetate was added and theorganic layer was washed with 0.1 N aqueous HCl (3×) and brine. Theorganic layer was dried over Na₂SO₄ and the solvent was removed underreduced pressure.

The residue was dissolved in HFIP (8 mL). After addition of TFA (200 μL)and TES (200 μL) the mixture was stirred for 30 min at RT. The solutionwas extracted with heptane (16 mL, 6×) and diluted with DCM (16 mL).Solution was extracted with water (16 mL, 3×). Combined water phaseswere back extracted with DCM (8 mL). The combined DCM phases wereevaporated under reduced pressure.

UPLC analysis revealed a 9/1 ratio of regioisomers 24a and 24b (column:Kinetex 100×2.1 mm, 1.7 μm XB-C18 silica, pore size 100 Å, PhenomonexLtd, Aschaffenburg, Germany; flow rate 0.25 mL/min; solvent A:water+0.05% TFA (v/v), solvent B: acetonitrile+0.04% TFA; gradient:30-58% B (10 min), 58% B isocratic (10 min), 58-80% B (5 min), 80-99% (5min), wavelength 280 nm).

The residue was taken up in acetonitrile/water and 24a was isolated byRP-HPLC (solvent A: H₂O+0.01% HCl, solvent B: MeCN+0.01% HCl, gradient:57-62% B over 15 min). Mixed fractions were subjected to repurification.Fractions containing pure 24a were combined and lyophilized.

Yield 24a: 98 mg (39%)

MS: m/z 660.3=[M+H]⁺ (MW calculated=659.9 g/mol).

Abbreviations

-   AcOH acetic acid-   AIB 2-Aminoisobutyric acid-   BnBr benzylbromide-   Boc tert-Butoxycarbonyl--   BSA N,O-Bis-(trimethylsilyl)-acetamide-   COMU    (1-Cyano-2-ethoxy-2-oxoethylidenaminooxy)dimethylamino-morpholino-carbenium    hexafluorophosphate

Abbreviations

-   d day-   DIPEA diisopropylethylamine-   DCM dichloromethane-   DMAP 4-(Dimethylamino)pyridine-   DMF N,N-Dimethylformamide-   Dmob 2,4-dimethoxybenzyl-   DMSO dimethyl sulfoxide-   Dpr 2,3-Diaminopropionic acid-   EDC N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide-   EDTA ethylenediamine tetraacetic acid disodium salt dihydrate-   EtOAc ethyl acetate-   eq equivalent-   h Hour-   HFIP 1,1,1,3,3,3-Hexafluoroisopropanol-   HPLC high performance liquid chromatography-   LC/MS mass spectrometry-coupled liquid chromatography-   Mal maleimido-   MeOH methanol-   MeCN acetonitrile-   min Minute-   Mmt 4-Methoxytriphenylmethyl-   mol. Molecular-   m/z Mass/charge-   NaOH Sodium hydroxide-   NHS N-hydroxysuccinimide-   PEG Polyethylene glycol-   Pfp Pentafluorophenyl-   PyBOB Benzotriazol-1-yl-oxytripyrrolidinophosphonium    hexafluorophosphate-   PP polypropylene

Abbreviations

-   RT room temperature-   RP reversed phase-   sat. saturated-   soln. solution-   T temperature-   T3P propyl phosphonic anhydride-   TCP 2-Chlorotrityl chloride resin-   TES Triethylsilane-   Trt Trityl-   Tmob 2,4,6-trimethoxybenzyl-   TMS trimethylsilyl-   TransCon transiently conjugated-   THF tetrahydrofuran-   TFA trifluoroacetic acid-   UPLC ultra performance liquid chromatography-   UV ultra violet

While this invention has been described in conjunction with the specificembodiments outlined above, it is evident that many alternatives,modifications, and variations will be apparent to those skilled in theart. Accordingly, the preferred embodiments of the invention as setforth above are intended to be illustrative, not limiting. Variouschanges may be made without departing from the spirit and scope of theinventions as defined in the following claims.

1. A method of treating, controlling, delaying, or preventing in amammalian patient in need of the treatment of one or more conditions,comprising: administering by inhalation to said patient a diagnosticallyand/or therapeutically effective amount of a carrier-linked treprostinilprodrug of formula (II), or a pharmaceutical salt thereof:

wherein each T is independently selected from structures (i) to (v):

wherein: dashed lines indicating attachment to the rest of the molecule;y is an integer ranging of from 1 to 64; R^(a1) is selected from thegroup consisting of: unsubstituted alkyl, substituted alkyl,unsubstituted phenyl, substituted phenyl, unsubstituted naphthyl,substituted naphthyl, unsubstituted indenyl, substituted indenyl,unsubstituted indanyl, substituted indanyl, unsubstituted tetralinyl,substituted tetralinyl, unsubstituted C₃₋₁₀ cycloalkyl, substitutedC₃₋₁₀ cycloalkyl, unsubstituted 4- to 7-membered heterocyclyl,substituted 4- to 7-membered heterocyclyl, unsubstituted 9- to11-membered heterobicyclyl, and substituted 9- to 11-memberedheterobicyclyl; R^(a2) is selected from the group consisting of: H,unsubstituted alkyl, and substituted alkyl; R^(a3) and R^(a4) areindependently selected from the group consisting of: H, unsubstitutedalkyl, and substituted alkyl; n is 0 or 1; Q is a spacer moiety;optionally, R^(a1) and R^(a3) are joined together with the atoms towhich they are attached to form a ring A; A is selected from the groupconsisting of: phenyl, naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀cycloalkyl, 4- to 7-membered aliphatic heterocyclyl, and 9- to11-membered aliphatic heterobicyclyl, wherein A is unsubstituted orsubstituted; and Z¹ is a carrier comprising a covalently bound polymer;and wherein R^(a2) and R^(a4) are absent if A is an aromatic ring. 2.The method of claim 1; wherein R^(a2) is H.
 3. The method of claim 1;wherein R^(a4) is selected from H, C₁₋₆ alkyl or substituted C₁₋₆ alkyl.4. The method of claim 1; wherein R^(a1) and R^(a3) are joined togetherwith the atoms to which they are attached to form a ring A; wherein A isselected from the group consisting of: phenyl; naphthyl, indenyl,indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 4- to 7-membered aliphaticheterocyclyl, and 9- to 11-membered aliphatic heterobicyclyl; andwherein A is unsubstituted or substituted.
 5. The method of claim 1;wherein T is selected from structure (iii).
 6. The method of claim 1;wherein y is 4, 6, 8, 10, or
 12. 7. The method of claim 1; wherein thecarrier-linked treprostinil prodrug has the structure of formula (II-A):

wherein: each T is independently selected from structures (i) to (v):

wherein: dashed lines indicating attachment to the rest of the molecule;y is an integer ranging of from 1 to 64; R^(a2) is selected from H,unsubstituted alkyl, and substituted alkyl; R^(a4) is selected from thegroup consisting of: H, unsubstituted alkyl, and substituted alkyl; A isselected from the group consisting of: phenyl, naphthyl, indenyl,indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 4- to 7-membered aliphaticheterocyclyl, and 9- to 11-membered aliphatic heterobicyclyl, wherein Ais unsubstituted or substituted; Q is a spacer moiety; and Z¹ is acarrier comprising a covalently bound polymer.
 8. The method of claim 1;wherein Q in formula (II) is selected from the group consisting of:COOR^(a9), OR^(a9), C(O)R^(a9), C(O)N(R^(a9)R^(a9a)),S(O)₂N(R^(a9)R^(a9a)), S(O)N(R^(a9)R^(a9a)), S(O)₂R^(a9), S(O)R^(a9),N(R^(a9))S(O)₂N(R^(a9a)R^(a9b)), SR^(a9), N(R^(a9)R^(a9a)), OC(O)R^(a9),N(R^(a9))C(O)R^(a9a), N(R^(a9))S(O)₂R^(a9a), N(R^(a9))S(O)R^(a9a),N(R^(a9))C(O)OR^(a9a), N(R^(a9))C(O)N(R^(a9a)R^(a9b)),OC(O)N(R^(a9)R^(a9a)), W; C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl;wherein W C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl are optionallysubstituted with one or more R^(a10), which are the same or different;wherein: C₁₋₅₀ alkyl; C₂₋₅₀ alkenyl; and C₂₋₅₀ alkynyl are optionallyinterrupted by one or more groups selected from the group consisting of:—W—, —C(O)O, —O—, —C(O)—, —C(O)N(R^(a11))—, —S(O)₂N(R^(a11))—,—S(O)N(R^(a11))—, —S(O)₂—; —S(O)—, —N(R^(a11))S(O)₂N(R^(a11a))—, —S—,—N(R^(a11))—, —OC(O)R^(a11), —N(R^(a11))C(O)—, —N(R^(a11))S(O)₂—,—N(R^(a11))S(O)—, —N(R^(a11))C(O)O—, —N(R^(a11))C(O)N(R^(a11a))—, and—OC(O)N(R^(a11)R^(a11a)); R^(a9), R^(a9a), and R^(a9b) are independentlyselected from the group consisting of: H, W, and C₁₋₅₀ alkyl, C₂₋₅₀alkenyl, and C₂₋₅₀ alkynyl; wherein W, C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, andC₂₋₅₀ alkynyl are optionally substituted with one or more R^(a10), whichare the same or different; wherein: C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, andC₂₋₅₀ alkynyl are optionally interrupted by one or more groups selectedfrom the group consisting of: W, —C(O)O—, —O—, —C(O)—, —C(O)N(R^(a11))—,—S(O)₂N(R^(a11))—, —S(O)N(R^(a11))—, —S(O)₂—, —S(O)—,—N(R^(a11))S(O)₂N(R^(a11a))—, —S—, —N(R^(a11))□, —OC(O)R^(a11),—N(R^(a11))C(O)—, —N(R^(a11))S(O)₂—, —N(R^(a11))S(O)—,—N(R^(a11))C(O)O—, —N(R^(a11))C(O)N(R^(a11a))—, andOC(O)N(R^(a11)R^(a11a)); W is selected from the group consisting of:phenyl, naphthyl, indenyl, indanyl, tetralinyl, C₃₋₁₀ cycloalkyl, 4- to7-membered heterocyclyl, and 9- to 11-membered heterobicyclyl; wherein Wis optionally substituted with one or more R^(a10), which are the sameor different; R^(a10) is selected from the group consisting of: halogen,CN, oxo (═O), COOR^(a12), OR^(a12), C(O)R^(a12), C(O)N(R^(a12)R^(a12a)),S(O)₂N(R^(a12)R^(a12a)), S(O)N(R^(a12)R^(12a)), S(O)₂R^(a12),S(O)R^(a12), N(R^(a12))S(O)₂N(R^(a12a)R^(a12b)), SR^(a12);N(R^(a12)R^(a12a)), NO₂, OC(O)R^(a12), N(R^(a12))C(O)R^(a12a),N(R^(a12))S(O)₂R^(a12a), N(R^(a12))S(O)R^(a12a),N(R^(a12))C(O)OR^(a12a), N(R^(a12))C(O)N(R^(a12a)R^(a12b)),OC(O)N(R^(a12)R^(a12a)), and C₁₋₆ alkyl; wherein C₁₋₆ alkyl isoptionally substituted with one or more halogen, which are the same ordifferent; and R^(a11), R^(a11a), R^(a12), R^(a12a), and R^(a12b) areindependently selected from the group consisting of: H, and C₁₋₆ alkyl;wherein C₁₋₆ alkyl is optionally substituted with one or more halogen,which are the same or different.
 9. The method of claim 8; wherein each-Q- is independently -Q^(1a)-Q¹-*, wherein the asterisk indicates theconnection to Z¹; wherein: Q^(1a) is a bond selected from the groupconsisting of: C(O)O—, —O—, —C(O)—, —C(O)N(R^(a9a))—, —S(O)₂N(R^(a9a))—,—S(O)N(R^(a9a))—, —S(O)₂—, —S(O)—, —N(R^(a9a))O(O)₂N(R^(a9b))—, —S—,—N(R^(a9a))—, —OC(O)—, —N(R^(a9a))C(O)—, —N(R^(a9a))S(O)₂—,—N(R^(a9a))S(O)—, —N(R^(a9a))C(O)O—, N(R^(a9a))C(O)N(R^(a9b))—,—OC(O)N(R^(a9a))—, and —W—. Q¹ is selected from the group consisting of:C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl which are optionallysubstituted with one or more R^(a10), which are optionally interrupted,provided that Q¹ is at least C₂, by one or more groups selected from thegroup consisting of: C₃₋₇ cycloalkyl, 4- to 7-membered heterocyclyl,

wherein each of said group may individually be present one or moretimes; and which C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl mayoptionally be terminated at the end connected to Z¹ by a group selectedfrom C₃₋₇ cycloalkyl, 4- to 7-membered heterocyclyl,

wherein: R^(a9a) is as defined in claim 8; R^(a10) is selected from thegroup consisting of: halogen, CN, oxo (═O), COOR^(a12), OR^(a12),C(O)R^(a12), C(O)N(R^(a12)R^(a12a)), S(O)₂N(R^(a12)R^(a12)),S(O)N(R^(a12)R^(a12a)), S(O)₂R^(a12), S(O)R^(a12),N(R^(a12))S(O)₂N(R^(a12a)R^(a12b)), SR^(a12), N(R^(a12)R^(a12a)), NO₂,OC(O)R^(a12), N(R^(a12))C(O)R^(a12a), N(R^(a12))S(O)₂R^(a12a),N(R^(a12))S(O)R^(a12a), N(R^(a12))C(O)OR^(a12a),N(R^(a12))C(O)N(R^(a12a)R^(a12b)), OC(O)N(R^(a12)R^(a12a)), and C₁₋₆alkyl; wherein C₁₋₆ alkyl is optionally substituted with one or morehalogen, which are the same or different; R^(a12), R^(a12a) and R^(a12b)are independently selected from the group consisting of: H; and C₁₋₆alkyl; wherein C₁₋₆ alkyl is optionally substituted with one or morehalogen, which are the same or different; and R¹³ and R^(13a) areindependently selected from the group consisting of: H, C₁₋₆ alkyl, C₂₋₆alkenyl, and C₂₋₆ alkynyl.
 10. The method of claim 1; wherein thecarrier-linked treprostinil prodrug is of formula (IIaa):

wherein: each T is independently selected from structures (i) or (iii):

wherein: dashed lines indicate attachment to the rest of the molecule; yis an integer ranging of from 1 to 64; R^(a2) is selected from H,unsubstituted alkyl, and substituted alkyl; R^(a4) is selected from H,unsubstituted alkyl, and substituted alkyl; ring A¹ is a C₃₋₁₀cycloalkyl; 4- to 7-membered aliphatic heterocyclyl; or 9- to11-membered aliphatic heterobicyclyl; wherein A¹ is unsubstituted orsubstituted; Q¹ is selected from C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl which are optionally substituted with one or more R^(a10), whichare optionally interrupted, provided that Q¹ is at least C₂, by one ormore groups selected from the group consisting of: C₃₋₇ cycloalkyl, 4-to 7-membered heterocyclyl,

wherein each of said group may individually be present one or moretimes; and which C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₁₋₅₀ alkynyl mayoptionally be terminated at the end connected to Z¹ by a group selectedfrom C₃₋₇ cycloalkyl, 4- to 7-membered heterocyclyl,

wherein: R^(a10) is selected from the group consisting of: halogen, CN,oxo (═O), COOR^(a12), OR^(a12), C(O)R^(a12), C(O)N(R^(a12)R^(a12a)),S(O)₂N(R^(a12)R^(a12a)), S(O)N(R^(a12)R^(a12a)), S(O)₂R^(a12),S(O)R^(a12), N(R^(a12))S(O)₂N(R^(a12a)R^(a12b)), SR^(a12),N(R^(a12)R^(a12a)), NO₂, OC(O)R^(a12), N(R^(a12))C(O)R^(a12a),N(R^(a12))S(O)₂R^(a12a), N(R^(a12))S(O)R^(a12a),N(R^(a12))C(O)OR^(a12a), N(R^(a12))C(O)N(R^(a12a)R^(a12b)),OC(O)N(R^(a12)R^(a12a)), and C₁₋₆ alkyl; wherein C₁₋₆ alkyl isoptionally substituted with one or more halogen, which are the same ordifferent; R^(a12), R^(a12a) and R^(a12b) are independently selectedfrom the group consisting of: H, and C₁₋₆ alkyl; wherein C₁₋₆ alkyl isoptionally substituted with one or more halogen, which are the same ordifferent; R¹³ and R^(13a) are independently selected from H, C₁₋₆alkyl, C₂₋₆ alkenyl, or C₂₋₆ alkynyl; and Z¹ is a carrier comprising acovalently bound polymer.
 11. The method of claim 1; wherein thecarrier-linked treprostinil prodrug is of formula (IIab):

wherein: each T is independently selected from structures (i) or (iii):

wherein: dashed lines indicate attachment to the rest of the molecule; yis an integer ranging of from 1 to 64; R^(a2) is selected from H,unsubstituted alkyl, and substituted alkyl; R^(a4) is selected from H,unsubstituted alkyl, and substituted alkyl; ring A¹ is a C₃₋₁₀cycloalkyl; 4- to 7-membered aliphatic heterocyclyl; or 9- to11-membered aliphatic heterobicyclyl; wherein A¹ is unsubstituted orsubstituted; Q¹ is selected from C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀alkynyl which are optionally substituted with one or more R^(a10), whichare optionally interrupted, provided that Q¹ is at least C₂, by one ormore groups selected from the group consisting of: C₃₋₇ cycloalkyl, 4-to 7-membered heterocyclyl,

wherein each of said group may individually be present one or moretimes; and which C₁₋₅₀ alkyl, C₂₋₅₀ alkenyl, and C₂₋₅₀ alkynyl mayoptionally be terminated at the end connected to Z¹ by a group selectedfrom C₃₋₇ cycloalkyl, 4- to 7-membered heterocyclyl,

wherein: R^(a10) is selected from the group consisting of: halogen, CN,oxo (═O), COOR^(a12), OR^(a12), C(O)R^(a12), C(O)N(R^(a12)R^(a12a)),S(O)₂N(R^(a12)R^(a12a)), S(O)N(R^(a12)R^(a12a)), S(O)₂R^(a12),S(O)R^(a12), N(R^(a12))S(O)₂N(R^(a12a)R^(a12b)), SR^(a12),N(R^(a12)R^(a12a)), NO₂, OC(O)R^(a12), N(R^(a12))C(O)R^(a12a),N(R^(a12))S(O)₂R^(a12a), N(R^(a12))S(O)R^(a12a),N(R^(a12))C(O)OR^(a12a), N(R^(a12))C(O)N(R^(a12a)R^(a12b)),OC(O)N(R^(a12)R^(a12a)), and C₁₋₆ alkyl; wherein C₁₋₆ alkyl isoptionally substituted with one or more halogen, which are the same ordifferent; R^(a12), R^(a12a) and R^(a12b) are independently selectedfrom the group consisting of: H, and C₁₋₆ alkyl; wherein C₁₋₆ alkyl isoptionally substituted with one or more halogen, which are the same ordifferent; R¹³ and R^(13a) are independently selected from H, C₁₋₆alkyl, C₂₋₆ alkenyl, or C₂₋₆ alkynyl; and Z¹ is a carrier comprising acovalently bound polymer.
 12. The method of claim 1; wherein thecarrier-linked treprostinil prodrug is of formula (IIac) or (IIad):

wherein: each T is independently selected from structures (i) or (iii):

Z¹ is a carrier comprising a covalently bound polymer; y is an integerranging of from 1 to 64; x is selected from 2, 3, 4, 5, 6, 7 or 8; andX₁ is selected from C₁₋₁₅ alkyl, C₂₋₁₅ alkenyl and C₂₋₁₅ alkynyl, whichare optionally substituted or interrupted by one or more groups selectedfrom the group consisting of: C₃₋₇ cycloalkyl, 4- to 7-memberedheterocyclyl,

wherein R¹³ and R^(13a) are independently selected from H, C₁₋₆ alkyl,C₂₋₆ alkenyl, or C₂₋₆ alkynyl.
 13. The method of claim 1; wherein thecarrier-linked treprostinil prodrug is of formula (IIb):

wherein: X₁ is selected from C₁₋₁₅ alkyl, C₂₋₁₅ alkenyl and C₂₋₁₅alkynyl, which are optionally substituted or interrupted by one or moregroups selected from the group consisting of: C₃₋₇ cycloalkyl, 4- to7-membered heterocyclyl,

wherein: R¹³ and R^(13a) are independently selected from H, C₁₋₆ alkyl,C₂₋₆ alkenyl, or C₂₋₆ alkynyl; y is an integer ranging of from 1 to 64;x is selected from 2, 3, 4, 5, 6, 7 or 8; and Z¹ is a carrier comprisinga covalently bound polymer.
 14. The method of claim 12; wherein x is 6.15. The method of claim 1; wherein the carrier-linked treprostinilprodrug has the structure of formula (IIba):

wherein: y is an integer ranging of from 1 to 64; and Z¹ is a carriercomprising a covalently bound polymer.
 16. The method of claim 1;wherein the carrier Z¹ has the structure of formula (VII):BA)_(n)  (VII); wherein: B is branching core; A is a poly(ethyleneglycol)-based polymeric chain; and n is an integer of from 3 to
 32. 17.The method of claim 1; wherein Z¹ represents a moiety (IIca):

wherein: t ranges from 80 to 160; w ranges from 2 to 6; and dashed linesindicate attachment to the rest of the carrier-linked treprostinilprodrug.
 18. The method of claim 17; wherein w is 2 or
 3. 19. The methodof claim 1; wherein the carrier-linked treprostinil prodrug is offormula (IIc), or a pharmaceutically acceptable salt thereof:

wherein y is 4 and Z¹ represents a moiety (IIca):

wherein: dashed lines indicate attachment to the rest of the structureof formula (IIc); t ranges from 80 to 160; and w is 2 or
 3. 20. Themethod of claim 1; wherein R^(a1) is C₁₋₆ alkyl or substituted C₁₋₆alkyl.
 21. The method of claim 1; wherein R^(a3) is H, C₁₋₆ alkyl orsubstituted C₁₋₆ alkyl.
 22. The method of claim 1; wherein thecarrier-linked treprostinil prodrug, or a pharmaceutically acceptablesalt thereof, as claimed in claim 1 is administered as part of apharmaceutical composition that optionally further comprises one or morepharmaceutically acceptable excipients.
 23. The method of claim 22; (i)wherein the carrier-linked treprostinil prodrug is sufficiently dosed inthe pharmaceutical composition to provide a therapeutically effectiveamount of treprostinil for at least 12 hours in one application; and/or(ii) wherein a single dose of the pharmaceutical composition comprisesabout 2 to about 6 mg the treprostinil moiety T.
 24. The method of claim1; wherein the disease or disorder is pulmonary arterial hypertension.25. The method of claim 1; wherein R^(a1) and R^(a3) are joined togetherwith the atoms to which they are attached to form a ring A; and whereinA is selected from the group consisting of: cyclopropane, cyclobutane,cyclopentane, cyclohexane, and cycloheptane.